Method for predicting ecdysis of blue crab based on behavior and morphological characteristics

Abstract

The application discloses a method for predicting the molting of blue crabs based on behavior and morphological characteristics, which comprises the identification of the pre-molting stage and the imminent molting stage, the determination of the pre-molting stage of 12-15 days by meeting at least three of the five quantitative characteristics of the pre-molting stage for more than 3 days, the determination of the imminent molting stage within 1-2 days by meeting four characteristics of the imminent molting stage, and the supporting grading control measures. The method for predicting the molting of blue crabs based on behavior and morphological characteristics has the advantages that the quantitative characteristics are measured by standardized instruments, the prediction accuracy reaches 97.5%, which is 66.7% higher than 58.5% of the traditional method, the molting success rate can reach 96.0%, the mortality rate is reduced to 4.0%, the early warning window is prolonged by more than 500%, and the method is suitable for various blue crabs, which is beneficial to the improvement of the initiative and benefits of breeding management.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to a method for predicting the molting of mud crabs based on behavioral and morphological characteristics. Background Technology

[0002] Mud crabs, an important economic crustacean in southeastern coastal my country, encompass several species including the mud crab (Scylla serrata) and the mud crab (Scylla serratedensis). Their meat is delicious and nutritious, possessing both edible and economic value, making them a core category in aquaculture. Molting is a crucial physiological process in the growth and development of mud crabs, spanning their entire life cycle. Through molting, mud crabs achieve body growth, shell renewal, and damage repair; the success rate of molting directly determines the survival rate and yield of farmed crabs. However, the molting period of mud crabs is extremely sensitive to the external environment, easily affected by factors such as water quality, feed, and predators. If the molting timing is not accurately predicted and targeted management measures are not taken, problems such as molting failure, cannibalism, or disease infection are highly likely to occur, causing significant economic losses to the aquaculture industry.

[0003] Predicting molting in mud crabs faces several key challenges: First, the molting process is regulated by multiple factors, including physiological rhythms and environmental parameters, and the molting cycle fluctuates significantly due to different growth stages and individual differences. Second, physiological changes in the early and near stages of molting are often subtle, making it difficult to detect minute morphological and behavioral changes with traditional visual observation. Third, the dynamic nature of the aquaculture environment (such as fluctuations in water temperature and salinity) further increases the uncertainty in predicting molting timing.

[0004] Currently, molting prediction technologies in the industry are mainly divided into three categories: The first is the experience-based judgment method, which relies on farmers to predict molting by observing macroscopic characteristics such as the crab's body color and feeding activity. This method is heavily influenced by subjective experience, has low accuracy, and cannot provide early warnings. The second is the single-indicator monitoring method, which predicts molting by monitoring water temperature, salinity, or a single morphological indicator (such as shell hardness). This method ignores the coordinated changes in the crab's physiology and behavior, resulting in a high misjudgment rate. The third is the instrument monitoring method, which uses water quality monitors and imaging equipment to monitor single parameters. While this improves data objectivity, it lacks a multi-indicator correlation model, making it difficult to comprehensively reflect the molting physiological mechanism and resulting in insufficient timeliness of early warnings. All of these methods suffer from low accuracy, delayed warnings, or narrow applicability, failing to meet the needs of large-scale aquaculture for precise management during the molting period.

[0005] This invention addresses the shortcomings of existing technologies by proposing a method for predicting the molting of mud crabs based on behavioral and morphological characteristics. By integrating and quantifying multi-dimensional indicators, it solves the core problems of low accuracy and poor timeliness in traditional methods, providing technical support for the refined management of mud crab molting. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the purpose of this invention is to provide a method for predicting the molting of mud crabs based on behavioral and morphological characteristics. The method of this invention can determine the pre-molting stage (12-15 days before molting) and the imminent molting stage (1-2 days before molting). Molting in mud crabs is a complex physiological and metabolic process that requires a complete cycle of "energy accumulation—new shell construction—old shell shedding": 12-15 days before molting, to meet the energy requirements for new shell synthesis and body growth, mud crabs will actively increase their food intake and prefer high-protein feed. Simultaneously, hemolymph accumulates in the appendage joints, causing joint swelling and discoloration. The cephalothorax develops convex edges due to the development of the new shell, and the abdominal umbilicus... As the crab undergoes physiological changes, its bristles grow longer and gradually change color. The abdomen bulges due to water accumulation and the new shell taking up space. One to two days before molting, the crab's physiological state undergoes a dramatic shift. It first accumulates energy by lying still, then, as the old shell loosens and the new shell is about to break through, its activity level increases significantly, and its appendages stand upright to find support for molting. Certain areas of the abdominal carapace become less hard due to the redissolution of calcium carbonate from the old shell, and the respiratory rate increases to meet the demands of high metabolism. Simultaneously, the crab cleans its body surface of attached substances and loosens the seams of the old shell, making final preparations for molting. Quantifying and combining these key physiological signals throughout the molting cycle avoids misjudgments caused by environmental fluctuations or individual differences in a single characteristic. Furthermore, by setting a "multiple feature satisfaction + duration" framework, it filters out accidental changes, achieving precise positioning of the molting stage.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for predicting the molting period of a mud crab, the molting period including a pre-molting period and a near-molting period; the method includes the following steps: acquiring behavioral and morphological data of the mud crab, and predicting the pre-molting and near-molting periods of the mud crab; the behavioral data includes the mud crab's feeding behavior, activity frequency, respiratory frequency, and wiping behavior; the morphological data includes the mud crab's appendage joint characteristics, cephalothorax edge characteristics, abdominal umbilical setae length, abdominal umbilical color, abdominal morphological changes, and carapace hardness.

[0008] In some embodiments of the present invention, the abdominal morphological changes include changes in the vertical distance between the central portion of the plastron and the posterior edge of the cephalothorax.

[0009] In some embodiments of the present invention, the wiping behavior refers to the action of the mud crab scraping the seam between the abdominal carapace and the carapace with its claws and the accompanying action of its walking legs cleaning the surface of the crab.

[0010] In some embodiments of the present invention, the prediction of the pre-molting period is based on the feeding behavior of the mud crab, appendage joint characteristics, cephalothorax edge characteristics, length of the umbilical setae, color of the umbilical setae, and changes in abdominal morphology; the pre-molting period of the mud crab is 12-15 days before molting.

[0011] In some embodiments of the present invention, when the mud crab satisfies at least 3 of the following (1)-(5), it is predicted that the mud crab is entering the pre-molting stage; (1) The daily food intake of mud crabs is 20% or more higher than the normal baseline level, and they prioritize protein intake; the normal baseline level is the average daily food intake of mud crabs 7-20 days after molting; the protein includes at least one of fish, shrimp and shellfish. (2) The diameter of the limb joint increases by 3% or more compared to the non-molting period; at the same time, the epidermal color value at the joint decreases by 10% or more compared to the normal area; the normal area is the area adjacent to the joint. (3) The posterior and lateral margins of the cephalothorax of the blue crab form continuous raised lines, and the width of the raised lines is greater than or equal to 0.5 mm; (4) The length of the bristles on the outer edge of the abdomen of the mud crab increases by 20% or more compared with the non-molting period, when the color value of the abdomen navel L is ≥80 and the b value is 20-30; the color values ​​of the abdomen navel L and b are obtained using the CIELab color space. (5) The vertical distance between the central part of the plastron and the posterior edge of the cephalothorax increases by 5% or more compared to the non-molting period when the body width is similar; The non-molting period is 7-15 days after molting.

[0012] In some embodiments of the present invention, the protein includes feed with a protein content of ≥40%.

[0013] In some embodiments of the present invention, the non-molting period is the stable period of molting in mud crabs; the daily food intake of mud crabs during the non-molting period fluctuates by no more than ±5%.

[0014] In some embodiments of the present invention, the preferred protein intake includes the fact that mud crabs prioritize feeding on fish, shrimp, and shellfish over other feeds such as organic debris and low-protein algae.

[0015] In some embodiments of the present invention, the joint diameter is measured using an electronic caliper with an accuracy of 0.01 mm. The measurement sites are the junction of the coxae and somites of the chelipeds and the junction of the coxae and somites of the walking legs. Each site is measured three times, and the average value is compared with the average value of the corresponding site under normal conditions to calculate the swelling rate.

[0016] In some embodiments of the present invention, the colorimetric value is measured using a standard colorimetric card (GB / T 22290-2008) and compared under natural light conditions (light intensity 5000-10000 lx). The measurement area is the swollen area of ​​the limb joint and the adjacent normal skin area. Each area is measured 3 times and the average value is taken.

[0017] In some embodiments of the present invention, the width of the raised line is measured using a digital vernier caliper with an accuracy of 0.01 mm. The measurement positions are the midpoint of the posterior edge of the cephalothorax and the midpoint of the left and right lateral edges, a total of 3 points. Each point is measured 3 times perpendicular to the direction of the line, and the arithmetic mean is taken as the final width value.

[0018] In some embodiments of the present invention, the bristle length is measured by a high-precision image measuring instrument, including the following steps: fixing the crab sample on the stage, acquiring a high-definition image of the bristle area at the outer edge of the abdominal navel, converting the pixels to the actual length using a standard calibration ruler, and measuring the length along the bristle main axis with a measurement accuracy of ±0.1 mm.

[0019] In some embodiments of the present invention, the method for measuring the color of the umbilicus is as follows: an image of the umbilicus region is acquired under a standard light source, and the L value and b value are extracted using the CIE Lab color space; when L≥80 and the b value is in the range of 10-20, it is determined to be pure white, and when the b value rises to the range of 20-30, it is determined to be a gradual change towards pale yellow.

[0020] In some embodiments of the present invention, the standard light source is obtained using a standard light source box.

[0021] In some embodiments of the present invention, the vertical distance between the central part of the abdominal carapace and the posterior edge of the cephalothorax is measured using a digital height gauge with an accuracy of 0.01 mm. During the measurement, the crab is placed on a horizontal experimental table with its abdominal carapace facing down and left to stand naturally. The vertical distance between the highest point of the central part of the abdominal carapace and the lowest point of the posterior edge of the cephalothorax is measured.

[0022] In some embodiments of the present invention, the molting approach period is used to predict the molting approach period of mud crabs based on activity frequency, shell hardness, respiratory frequency, and wiping behavior; the molting approach period is 1-2 days before the molting of mud crabs.

[0023] In some embodiments of the present invention, the shell hardness includes the hardness of the abdominal carapace region below the longest serrations on both sides of the cephalothorax of the blue crab.

[0024] In some embodiments of the present invention, when the mud crab enters the pre-molting stage and satisfies all of the following characteristics (1)-(4), it is predicted that the mud crab is entering the near-molting stage. (1) After the mud crab has been lying still for 24-48 hours, the number of times it moves per hour is 300% or more higher than during the lying-still period; and the crab appears in an upright position with its appendages at least 5 times per hour, each time lasting at least 15 seconds. (2) The hardness of the abdominal plate below the longest serration on both sides of the cephalothorax of the mud crab is 20% or more lower than that of individuals not in the molting period; (3) The number of times the gill covers open and close is counted. The breathing rate of the blue crab reaches 13-16 times per minute. (4) The number of times the mud crab scrapes the seam between the abdominal carapace and the carapace with its claws shall not be less than 20 times per hour, each time lasting not less than 10 seconds, accompanied by the cleaning action of the walking legs on the surface of the body, and the total daily wiping time shall not be less than 5 minutes.

[0025] In some embodiments of the present invention, the hardness of the plastron is measured using a Shore hardness tester (Type A). The instrument is calibrated before measurement. During measurement, the probe is perpendicular to the plastron surface, and a pressure of 1N is applied. Each measurement site is measured 3 times, and the average value is compared with the average hardness of the same site of individuals not in the molting period to calculate the hardness reduction rate.

[0026] In some embodiments of the present invention, the upright position of the appendages is characterized by the walking legs or chelipeds vertically supporting the body and the head and thorax being raised off the ground.

[0027] In some embodiments of the present invention, the activity frequency, the number of times the appendages are erected, and the number of digging and wiping actions are continuously recorded by a high-definition camera (frame rate ≥ 30 fps) for a duration of not less than 1 hour, and the relevant behavioral parameters are statistically analyzed using image analysis software; the respiratory rate is determined by manually counting the number of times the gill covers open and close, with each count lasting 1 minute, and the count is repeated 3 times at 5-minute intervals, and the average value is taken.

[0028] In some embodiments of the present invention, the non-molting mud crab is a healthy mud crab whose body width difference is ≤5% and weight difference is ≤10% compared with the mud crab to be tested, and which is in a stable period of 7-15 days after molting.

[0029] In some embodiments of the present invention, the mud crab includes mud crabs such as mud crab, mud crab, mud crab, mud crab, or hybrid individuals thereof.

[0030] A second aspect of the present invention provides a method for raising mud crabs in the pre-molting stage, the method comprising the following steps: after predicting the pre-molting stage using the method described above, performing any one or more of the following steps (1)-(5): (1) Increase the ambient shading rate to 60-70%; (2) Increase the calcium ion concentration in the water to 400-500 mg / kg and maintain dissolved oxygen at 5.5-6.0 mg / L; (3) Increase the number of feedings to 3-5 times, with a single feeding amount of 3-4% of the body weight of the crab. The feeding includes feeding with feed from at least one source of fish, shrimp and shellfish. (4) Adjust the light intensity to 500-1000 lx; (5) Control the ammonia nitrogen in the water body to ≤0.1mg / L and the nitrite to ≤0.05mg / L.

[0031] A third aspect of the present invention provides a method for raising mud crabs near the molting stage, the method comprising the following steps: after predicting the near molting stage using the method described above, performing any one or more of the following steps (1)-(6): (1) Raise the water temperature to 26-28℃; (2) Adjust the light intensity to 100-200 lx; (3) Adjust the photoperiod to 8-10 hours of light and 14-16 hours of darkness; (4) Add 0.5-1 g / m³ to the water body according to the ratio of usage to water volume. 3 Vitamin C at 0.1-0.5g / m 3 EDTA-2Na; (5) Adjust the dissolved oxygen in the water to 6.5-7.5 mg / L; (6) Stop feeding.

[0032] In some embodiments of the present invention, raising the water temperature to 26-28°C includes raising the water temperature by 2-3°C at a heating rate of 0.2°C-0.4°C per hour, and finally stabilizing it at 26-28°C.

[0033] The beneficial effects of this invention are: This invention provides a method for predicting the molting of mud crabs based on behavioral and morphological features, which has significant advantages over traditional techniques, as detailed below: I. The indicator system is comprehensive, simultaneously monitoring limb joint swelling and discoloration, convexity of the cephalothorax edge, slight bulging of the plastron, softening of tissues in specific areas, increased respiratory rate, and behavioral indicators such as wiping, covering the complete physiological change chain from the pre-molting stage to the imminent stage; abandoning the traditional single indicator or visual observation method of judgment; II. Quantitative Judgment Criteria: Quantitative indicators such as feeding behavior analysis, standard colorimetric cards, width values, swelling rates, and gill opening and closing frequency replace subjective judgment, improving prediction accuracy. All characteristics are measured using standardized tools such as 0.01mm calipers and the GB / T22290-2008 standard colorimetric card. In aquaculture tests, monitoring of 400 individuals of *Scylla serrata* and *Scylla serrulata* showed that the accuracy rate of this invention's pre-molting warning (12-15 days) reached 97.9%, the accuracy rate of the near-molting period (1-2 days) reached 97.1%, and the overall prediction accuracy reached 97.5%. The misjudgment rate decreased from 41.5% to 2.5% compared to the control group. The synergistic verification of multiple indicators effectively avoids judgment errors caused by environmental fluctuations and individual differences.

[0034] Third, the early warning system is clearly tiered, distinguishing between "pre-molting" warnings and "imminent molting" confirmations to adapt to the management needs of different stages, significantly improving molting success rates and reducing mortality rates. This invention constructs a tiered early warning system for pre-molting and impending molting confirmations, along with targeted management measures. In aquaculture trials, the experimental group (n=100) using this invention achieved a molting success rate of 95.0%, while the control group (n=100) using traditional management only achieved 68.0%. The concealed structure reduces cannibalism during molting, and precise water quality control supports new shell formation, reducing the molting mortality rate from 32.0% to 4.0%, effectively solving the core problems of difficult molting and high mortality rates in traditional aquaculture. This invention's method for predicting molting in mud crabs based on behavioral and morphological characteristics significantly extends the early warning management window, substantially improving the initiative in aquaculture management. Existing technologies can only predict molting when mud crabs show obvious abnormalities such as ceasing feeding (1-2 days before molting), leading to rushed management operations. This invention monitors early, subtle morphological changes in the mud crab, such as the joint diameter of the basal joints of its chelipeds and walking legs, changes in the skin color at the joints, the width of the continuous raised lines forming on the posterior and lateral edges of the cephalothorax, the length of the bristles on the outer edge of the abdominal navel, and the color of the abdominal navel. It can provide early warnings 12-15 days before molting. Compared to the traditional 1-2 day warning period, the effective warning management window of this invention is extended by more than 500%, allowing farmers sufficient time to complete tasks such as water quality adjustment, setting up hiding places, and preparing high-protein feed (protein content ≥40%), thereby improving the implementation rate of management measures and reversing the passive situation in aquaculture management.

[0035] IV. High operability: Utilizing conventional equipment such as high-precision image measuring instruments and high-definition cameras, this invention requires no complex hardware investment, facilitating large-scale promotion. The method for predicting molting in mud crabs based on behavioral and morphological characteristics reduces aquaculture management costs and significantly improves overall economic benefits. This invention uses continuous shooting with a high-definition camera at a frame rate ≥30fps, combined with image analysis software, to automatically collect behavioral parameters such as activity frequency and the number of times appendages are upright, replacing the traditional manual inspections conducted by aquaculture personnel every 2 hours. Simultaneously, standardized instrument measurement reduces repetitive manual judgment, reducing the frequency of manual inspections to twice per day. Furthermore, targeted feeding supported by accurate prediction (feeding 3 times daily during the early molting stage, with each feeding amount being 3%-4% of body weight) increases feed utilization from 52.5% to 76.8% (an increase of 24.3 percentage points). Combined with the advantage of increasing the overall molting survival rate of mud crabs from the traditional 82.0% to 98.0%, the overall aquaculture economic benefits are significantly improved. Detailed Implementation

[0036] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art. The equipment used in the embodiments is shown in Table 1 below: Table 1 Experimental Instruments and Equipment

[0037] Example 1 This embodiment provides a screening method for predicting the molting behavior and morphological characteristics of mud crabs.

[0038] In this embodiment, enhanced feeding behavior, changes in appendage joints, cephalothorax edge features, changes in the length and color of the umbilicus bristles, and changes in abdominal morphology were obtained through screening to determine that the animal had entered the "pre-molting" stage, which is 12-15 days before molting. Changes in feeding and activity patterns, softening of tissues in specific areas, increased respiratory rate, and wiping behavior were used to determine that the animal was approaching the molting stage and was expected to molt within 1-2 days.

[0039] The principle behind using enhanced feeding behavior traits to predict molting behavior lies in the fact that protein is the core raw material for building a new exoskeleton and repairing body tissues during the molting process of mud crabs. The main components of the new shell are chitin and protein complexes. Before molting, mud crabs need to accumulate a large amount of protein to meet the needs of new shell synthesis; otherwise, insufficient nutrition will lead to a soft new shell, difficulty in molting, or increased mortality after molting. Live bait such as small fish, shrimp, and shellfish with a protein content of ≥40% not only have a much higher protein content than organic debris and low-protein algae, but also have an amino acid composition that is closer to the nutritional needs of mud crabs, making them easier to digest and absorb, and quickly converting them into raw materials for building a new shell. At the same time, as carnivorous crustaceans, mud crabs naturally have a preference for live bait, and their feeding priority is higher than other types of bait. When this type of bait is provided, mud crabs have higher feeding enthusiasm and more stable feeding amounts, ensuring sufficient nutrient intake. If low-protein or indigestible feed is provided, even if the amount of food consumed is increased, it will be difficult to meet the nutritional needs required for molting. In fact, the low feed utilization rate will lead to water quality deterioration.

[0040] Meanwhile, the average daily feed intake of mud crabs during the stable period after molting (7-20 days post-molting) was used as a normal baseline. During this stable period, the daily feed intake of mud crabs fluctuated by no more than ±5%. After molting, mud crabs go through a growth cycle of "recovery period - stable period - molting preparation period": In the first 7 days after molting, the new shell of the mud crab has not yet fully hardened, and its physiological functions are in a recovery state, resulting in lower and more volatile feed intake; while in the 7-20 days after molting, the new shell has hardened, the physiological state of the mud crab is stable, its growth and metabolism are slow, and its feeding behavior is not affected by the physiological changes related to molting. The daily feed intake remains at a relatively constant level, with fluctuations controlled within ±5%, which provides stability and reliability as a normal baseline.

[0041] The characteristics of appendage joint changes include joint swelling and discoloration. For the swollen appendage joint changes, measurements were taken using electronic calipers with an accuracy of 0.01 mm. The diameter of the appendage joints at the junction of the chelipede coxae and somatic segments, and the junction of the walking legs coxae and somatic segments, was measured three times at each location. The average value was compared with the average value of the corresponding location under normal conditions to calculate the swelling rate. The principle is that during the pre-molting period, hormonal changes in the mud crab cause hemolymph to accumulate at the junction of the chelipede and walking legs coxae and somatic segments, providing nutrients and water for the development of the new shell. This leads to physiological swelling of the joints in these areas, with the joint diameter increasing by more than 3% compared to the normal state. This is an important morphological signal during the pre-molting period. Because the degree of swelling is quite subtle, ordinary measuring tools are difficult to accurately capture, and the swelling varies at different joint locations. Measuring only a single location may lead to data deviations due to individual posture or accidental factors. Using electronic calipers with an accuracy of 0.01mm allows for precise measurement of minute diameter changes, ensuring data accuracy. Measurements are taken at key joints of the chelipeds and walking legs to comprehensively reflect the overall swelling status of the crab's appendage joints. Measuring each area three times and averaging the results effectively reduces random errors during measurement, making the swelling rate calculation more objective. Insufficient precision of the measuring tools, unclear measurement locations, or too few measurements can lead to deviations in the swelling rate calculation, making it impossible to accurately determine whether the crab has entered the pre-molting stage. This standardized, high-precision measurement scheme for judging the degree of swelling in appendage joint changes converts joint swelling into quantitative swelling rate data, avoiding errors from subjective visual judgment and ensuring the consistency and reliability of this feature assessment, providing a solid quantitative basis for pre-molting identification.

[0042] The color change characteristics of the appendage joints were measured using a standard colorimetric card (GB / T22290-2008) under natural light conditions (light intensity 5000–10000 lx). The measurement area included the swollen area of ​​the appendage joint and the adjacent normal skin area. Each area was measured three times and the average value was taken. The principle is that in the early molting stage of the mud crab, due to the accumulation of hemolymph and epidermal aging, the epidermal cells gradually slough off, causing the epidermis at the joint to lighten in color, appearing whitish or milky white. The color value is more than 10% lower than that of the normal area. This color change is an important signal that the epidermis is about to separate from the old shell. However, color judgment is easily affected by lighting conditions and subjective visual differences of the observer. The color of the same part will appear significantly different under different light intensities, and it is difficult to accurately quantify the degree of color change by visual observation alone. Using a standard colorimetric card conforming to the national standard (GB / T 22290-2008), a unified color reference scale is provided, ensuring standardized colorimetric value judgment. Measurements are limited to natural light conditions of 5000-10000 lx to avoid interference from strong light, weak light, or artificial light sources, ensuring consistency in the measurement environment. Simultaneous measurement of swollen joint areas and adjacent normal skin areas, along with comparative calculation of the colorimetric value decrease rate, eliminates the influence of individual color differences in mud crabs, accurately capturing the unique color changes characteristic of the pre-molting stage. Three measurements are taken for each area, and the average value is used to reduce random errors and improve data reliability. Its function is to transform the subjective judgment indicator of limb joint color change into objective, quantifiable colorimetric value data, eliminating judgment biases caused by lighting and human factors, ensuring the accuracy and repeatability of limb joint change characteristics, and providing a scientific and unified judgment standard for pre-molting identification.

[0043] For the edge features of the cephalothorax, a digital vernier caliper with an accuracy of 0.01 mm was used to measure the width of the continuous raised lines formed on the posterior and lateral edges of the cephalothorax of the mud crab. Measurements were taken at three locations: the midpoint of the posterior edge of the cephalothorax and the midpoints of the left and right lateral edges. Each measurement was taken three times perpendicular to the line's direction, and the arithmetic mean was used as the final width value. The principle is that during the early molting stage of the mud crab, the new shell begins to develop and separate from the old shell. As the main protective structure of the body, the growth of the new shell inside the old shell causes obvious raised lines to appear on the posterior and lateral edges of the cephalothorax. The width of these lines directly reflects the degree of development of the new shell. When the width is ≥0.5 mm, it indicates that the new shell has reached a certain thickness, and the mud crab has entered the early molting stage. Because the raised lines on the edge of the cephalothorax may have local variations in thickness, measuring only a single location would result in unrepresentative data. Therefore, measurements were taken at three locations: the midpoint of the posterior edge of the cephalothorax and the midpoints of the left and right lateral edges. Since the line width is 0.5 mm or more, high-precision measuring tools are required for accurate measurement. Using a digital vernier caliper with an accuracy of 0.01mm, the system can meet the measurement needs for minute widths, ensuring data accuracy. Measurements are taken at three points: the midpoint of the posterior edge of the cephalothorax and the midpoints of the left and right lateral edges. This comprehensively reflects the overall width of the lines, avoiding misjudgments caused by local differences. Each measurement is taken three times perpendicular to the line's direction, and the arithmetic mean is calculated. This reduces measurement angle deviations and random errors, ensuring the objectivity and accuracy of the results. Random measurement positions, insufficient tool precision, or too few measurements can lead to inaccurate judgments of the raised line width, making it impossible to accurately identify pre-molting stages. This system provides a standardized, high-precision measurement method for cephalothorax edge features, using a raised line width ≥0.5mm as a quantitative indicator to avoid the ambiguity of subjective observation, ensuring the accuracy of this feature's judgment and providing crucial morphological evidence for pre-molting stage identification.

[0044] The length of the abdominal setae was measured using a high-precision image measuring instrument. The specific procedure involved fixing the mud crab sample on a stage, acquiring high-resolution images of the setae region around the outer edge of the abdominal setae, converting the pixel count to the actual length using a standard calibration ruler, and measuring the length along the main axis of the setae. The measurement accuracy was ±0.1 mm. The principle behind this is that the growth of the abdominal setae in mud crabs is closely related to their physiological cycle. During the pre-molting period, changes in hormone levels in the mud crab stimulate rapid growth of the abdominal setae, increasing by 20% or more compared to the non-molting period. This is an important physiological signal during the pre-molting period. However, the abdominal setae are thin and short, and the mud crabs may struggle during measurement. Directly using calipers or other tools can easily lead to setae breakage and measurement angle deviations, making it difficult to obtain accurate data. High-precision image measuring instruments perform non-contact measurements by acquiring high-definition images, avoiding damage to the bristles from direct measurement while clearly presenting the complete shape of the bristles. A standard calibration ruler accurately converts pixels to actual length, translating pixel distances in the image into actual physical lengths, ensuring measurement accuracy. Measurement along the bristle's main axis avoids length errors caused by measurement path deviation, achieving a measurement accuracy of ±0.1mm, sufficient to capture length increases of 20% or more. Improper measurement methods or insufficient tool precision can lead to distorted bristle length measurement data, making it impossible to accurately determine whether the characteristics of the pre-molting stage are met.

[0045] The method for measuring the color of the abdominal navel involves acquiring images of the navel area under a standard light source and extracting L and b values ​​using the CIE Lab color space. When L ≥ 80 and b value is within the range of 10-20, it is considered pure white. When the b value rises to the range of 20-30, it is considered a gradual change towards pale yellow. The principle behind this is that the color change of the mud crab's abdominal navel is closely related to its internal physiological state: during the non-molting period, the epidermal cells of the abdominal navel are metabolically stable and appear pure white; after entering the pre-molting stage, changes occur in the mud crab's water metabolism and pigment deposition, and the epidermis of the abdominal navel gradually changes towards pale yellow. This color change is an important signal of the pre-molting stage. However, subjective judgment of color is easily affected by differences in light source and observer's vision, making it difficult to establish a unified standard. The CIE Lab color space is an internationally recognized color quantification system. The L value represents brightness, and the b value represents the yellow-blue axis (the larger the b value, the more pronounced the yellow tint). This color space allows the color of the navel to be converted into objective numerical parameters. Acquiring images under standard light sources avoids interference from natural light intensity and color temperature variations, ensuring a consistent measurement environment. By setting clear L and b value ranges, "pure white" and "gradual transition to pale yellow" are quantified, transforming color judgment from subjective visual perception to objective data comparison. Without standardized measurement methods and quantification standards, judging navel color changes solely through visual observation is prone to bias, affecting the accuracy of early molting identification.

[0046] The abdominal morphological changes in mud crabs are characterized by a slight overall bulge in the abdomen. The vertical distance between the central part of the abdominal carapace and the posterior edge of the cephalothorax is increased compared to non-molting individuals with similar body width. Measurements were taken using a digital height gauge with an accuracy of 0.01 mm. During measurement, the mud crab was placed on a horizontal experimental table with its abdominal carapace facing down and allowed to rest naturally. The vertical distance between the highest point of the central part of the abdominal carapace and the lowest point of the posterior edge of the cephalothorax was measured. The principle is that in the pre-molting stage, mud crabs actively absorb water and accumulate it in the abdomen to provide space for the development of the new shell. Simultaneously, the growth of the new shell on the inner side of the abdomen causes an overall bulge, significantly increasing the vertical distance between the central part of the abdominal carapace and the posterior edge of the cephalothorax—by 5% or more compared to non-molting individuals with similar body width. This distance change directly quantifies the abdominal bulge characteristic. During the measurement process, the placement posture of the mud crab and the selection of the measurement reference point directly affect the measurement results: if the mud crab is not allowed to rest naturally, the abdomen may be in a contracted or twisted state due to struggling, leading to inaccurate distance measurements; if the measurement reference point is unclear, measurement position deviations will occur. Placing the mud crab on a horizontal experimental platform with its plastron facing down and allowing it to stand naturally ensures that the measurement reference plane is level and that the abdomen is in a natural state. Using a digital height gauge with an accuracy of 0.01 mm can accurately capture distance changes of up to 5%. Clearly measuring the vertical distance between the highest point in the center of the plastron and the lowest point at the posterior edge of the cephalothorax can avoid errors caused by fuzzy measurement points and ensure the consistency of measurement results.

[0047] The softening characteristics of specific tissues include a decrease in hardness of more than 20% in the abdominal carapace area below the longest serrations on both sides of the cephalothorax of the mud crab compared to non-molting individuals. This was measured using a Shore A hardness tester. The instrument was calibrated before measurement. During measurement, the probe was perpendicular to the abdominal carapace surface, and a pressure of 1 N was applied. Each measurement site was measured three times, and the average value was compared with the average hardness of the same site in non-molting individuals to calculate the rate of hardness reduction. The principle is that, near molting, the mud crab secretes molting hormones, which stimulate the epidermal cells on the inner side of the old shell to secrete enzymes that break down the proteins and calcium carbonate in the old shell, gradually softening and thinning it to facilitate molting. The specific area of ​​the abdominal carapace (below the longest serrations on both sides of the cephalothorax) is the main breakthrough point during molting, showing the most significant softening of the old shell, with a hardness decrease of more than 20% compared to non-molting individuals. This is a core morphological signal indicating the approaching molting stage. The accuracy of hardness measurement is affected by factors such as instrument calibration, measurement pressure, and probe angle. If the instrument is not calibrated, the measurement reference will be deviated; if the probe is not perpendicular or the applied pressure is unstable, the measurement data will fluctuate significantly. Calibrating the instrument before measurement ensures the accuracy of the hardness tester; applying a pressure of 1N perpendicular to the plastron surface ensures consistent measurement conditions and avoids distortion of hardness data due to differences in pressure or angle; taking three measurements for each part and averaging them reduces random errors and improves data reliability; comparing the hardness of the same part with that of individuals not in the molting period to calculate the rate of decrease can eliminate the hardness differences within the individual crab and accurately reflect the degree of softening of the old shell.

[0048] Activity pattern changes; increased respiratory rate and wiping behavior were recorded continuously using a high-definition camera (frame rate ≥30fps) for at least 1 hour, and relevant behavioral parameters were statistically analyzed using image analysis software. Respiratory rate was determined by manually counting the number of times the gill covers opened and closed, with each count lasting 1 minute, repeated 3 times at 5-minute intervals, and the average value was taken. One to two days before molting, the mud crab undergoes a dramatic physiological change. It first accumulates energy by lying still, then, due to the loosening of the old shell and the imminent emergence of the new shell, its activity rate increases significantly, and its appendages stand upright to find support for molting. Certain areas of the abdominal carapace experience a decrease in hardness due to the redissolution of calcium carbonate from the old shell, leading to an increased respiratory rate to meet the demands of high metabolism. Simultaneously, wiping behavior cleans the body surface of attached substances and loosens the seams of the old shell, making final preparations for molting.

[0049] Meanwhile, in this embodiment, healthy mud crabs (non-molting mud crabs) with a body width difference of ≤5% and a weight difference of ≤10% compared to the tested mud crabs, and which are in a stable period of 7-15 days after molting, were selected as the benchmark for measuring the characteristics of appendage joint changes, carapace edge characteristics, abdominal umbilical setae length and color changes, abdominal morphological changes, tissue softening characteristics in specific areas, increased respiratory rate, and wiping behavior. The principle is that the morphological parameters (such as appendage joint diameter and vertical distance between the abdominal carapace and carapace) and physiological parameters (such as abdominal carapace hardness and respiratory rate) of mud crabs are significantly affected by body width, weight, and growth stage: mud crabs with large differences in body width and weight have significant differences in their basic morphology and physiological indicators, and using them as a reference will lead to distorted comparison results; mud crabs within 7 days after molting have not fully hardened their new shells, their physiological state is unstable, and various parameters fluctuate greatly, making them unworthy of reference; and mud crabs in poor health may have abnormal morphological and physiological parameters due to disease, which cannot reflect the normal non-molting state.

[0050] Selecting individuals with a body width difference of ≤5% and a weight difference of ≤10% from the crab to be tested ensures that the reference crabs are similar in size to the crabs to be tested, eliminating the interference of body size differences on parameter comparison; limiting them to the stabilization period of 7-15 days after molting ensures that the physiological state of the reference crabs is stable and that all parameters are at normal levels; requiring healthy crabs avoids reference bias caused by disease factors.

[0051] This embodiment provides standardized quantitative measurement methods for characteristics such as enhanced feeding behavior, changes in appendage joints, cephalothorax edge features, changes in the length and color of the umbilicus setae, changes in abdominal morphology, changes in activity patterns, softening of tissues in specific areas, increased respiratory rate, and wiping behavior. This provides an objective and operable measurement basis for predicting molting in mud crabs and ensures the accuracy of the characteristic judgment.

[0052] Example 2 This embodiment provides a method for predicting the molting of mud crabs based on behavioral and morphological characteristics. The mud crabs in the non-molting period are healthy mud crabs with a body width difference of ≤5% and a weight difference of ≤10% compared to the mud crabs to be tested, and which are in a stable period of 7-15 days after molting. The culture water is filtered seawater with a salinity of 12-18%, aerated for 24 hours in advance, with the temperature stable at 24-26℃, the pH value of the water body of 7.8-8.2, and the dissolved oxygen content of ≥5.0mg / L, as detailed below.

[0053] The feeding behavior of mud crabs was recorded at fixed times every day (8:00, 14:00, 22:00), and the daily feed intake and preference for high-protein live bait were statistically analyzed.

[0054] The following five quantitative characteristics were measured daily using standardized instruments and monitored continuously for 15 days. The judgment criteria and duration of each characteristic were recorded. When the mud crab meets at least three of the five characteristics (1)-(5) and lasts for ≥3 days, it is marked as entering the pre-molting stage, which is 12-15 days away from molting.

[0055] (1) Enhanced feeding behavior Operating procedures: Feed a fixed amount of feed daily (initial feed amount is 5% of the body weight of the mud crab). After 2 hours, collect the remaining feed, dry it to constant weight, and calculate the actual feed intake. Record the feed intake for 7 consecutive days during the stabilization period, and take the average value as the normal baseline level. During the experiment, record the mud crab's feeding preference for high-protein live feed (fish (sardines), shrimp (Litopenaeus vannamei), and shellfish (razor clams) with protein content ≥40%, after being chopped and fed) and low-protein feed (corn, soybean meal). The normal baseline level is the average daily feed intake of mud crabs during the stabilization period after molting (7-20 days after molting), during which the daily feed intake of mud crabs fluctuates by no more than ±5%.

[0056] Daily food intake improvement rate = (daily food intake during the testing period - normal baseline level) / normal baseline level × 100%; The normal baseline level is the average daily feed intake of mud crabs during the stable period after molting (7-20 days after molting), during which the daily feed intake of mud crabs fluctuates by no more than ±5%.

[0057] Judgment criteria: If the daily food intake of mud crabs is 20% or more higher than the normal baseline level, and the feed is relatively low in protein, and the mud crabs preferentially consume high-protein fresh feed, then this characteristic criterion is met. (2) Characteristics of changes in appendiceal joints Operating steps: Use electronic calipers to measure the diameter at the junction of the coxae and somites of the chelipeds and the junction of the coxae and somites of the walking legs. Measure each location three times and take the average value. Use a standard colorimetric card under natural light conditions (light intensity 5000-10000 lx) to measure the color values ​​of the joint areas (junction of the coxae and somites of the chelipeds and the junction of the coxae and somites of the walking legs) and adjacent normal skin areas. Measure each area three times and take the average value. Compare the average value with the corresponding average value of the area under non-molting conditions to calculate the swelling rate.

[0058] Joint swelling rate = (joint diameter during the detection period - joint diameter before molting) / joint diameter in normal condition × 100%; Chromaticity value decrease rate = (Chromaticity value of adjacent normal area at joint site - Chromaticity value of joint site) / Chromaticity value of adjacent normal area × 100%; Judgment criteria: The base joints of the claws and walking legs of the mud crab show swelling due to hemolymph accumulation, with the joint diameter increasing by 3% or more compared to the normal state (joint swelling rate). At the same time, the skin at the joint becomes lighter in color, appearing whitish or milky white. Based on standard colorimetric card measurements, its color value is reduced by 10% or more compared to the normal area to meet the characteristics.

[0059] (3) Features of the cephalothorax margin Operating steps: Use a digital vernier caliper to measure the width of the raised lines at three points: the midpoint of the posterior edge of the cephalothorax and the midpoint of the left and right lateral edges. Measure each point three times perpendicular to the direction of the line and take the arithmetic mean.

[0060] Judgment criteria: The width of the raised line is ≥0.5mm to meet the feature.

[0061] (4) Characteristics of changes in the length of the umbilicus bristles and the color of the umbilicus Operating steps: Acquire high-resolution images of the bristles on the outer edge of the umbilicus using a high-precision image measuring instrument. Convert pixels to actual length using a standard calibration ruler and measure the length along the bristle's main axis. Acquire images of the umbilicus under a standard light source. Extract L-values ​​(brightness, ranging from 0 to 100, where 0 represents pure black and 100 represents pure white) and b-values ​​(ranging from yellow to blue, ranging from -128 to 127, where positive values ​​indicate a yellowish tint and negative values ​​indicate a bluish tint) using the CIELab color space. When the L-value is ≥80 and the b-value is in the range of 10-20, it is considered pure white. When the b-value rises to the range of 20-30, it is considered a gradual transition to pale yellow. A standard light source box is used to obtain the standard light source.

[0062] The growth rate of setae length = (setae length of mud crabs during the detection period - setae length of mud crabs outside the molting period) / setae length of mud crabs outside the molting period × 100%; Judgment criteria: The bristle length growth rate is ≥20%, and the color of the navel changes from pure white to light yellow, that is, the b value reaches 20-30 (light yellow gradient) to meet the characteristics.

[0063] (5) Characteristics of abdominal morphological changes Operating procedures: Place the mud crab on a horizontal experimental table with its plastron facing down and let it stand naturally. Use a digital height gauge to measure the vertical distance between the highest point in the center of the plastron and the lowest point at the posterior edge of the cephalothorax. Select mud crabs of similar body width that are not in their molting stage as a reference.

[0064] Vertical distance increase rate = (vertical distance during the detection period - vertical distance during the non-molting period) / vertical distance during the non-molting period × 100%; Judgment criterion: an increase rate of 5% or more is considered to meet the feature.

[0065] 4. The fourth day after the mud crab meets the characteristics of the pre-molting stage is recorded as the first day of the pre-molting stage. When the 10th day of the pre-molting stage begins, the behavior, hardness of the abdominal carapace and respiratory rate of the mud crab are continuously monitored. The monitoring is carried out 4 times a day (8:00, 12:00, 16:00 and 22:00). The behavior of the mud crab is continuously filmed with a high-definition camera for no less than 1 hour each time. The activity frequency, number of times the appendages stand upright and the number of times the wiping action are counted by image analysis software. When the mud crab meets all 4 characteristics of (1)-(4) below, it is determined that it has entered the "imminent molting period" stage and is expected to molt within 1-2 days. The activity frequency, number of times the appendages stand upright and the number of times the wiping action are counted by continuous filming with a high-definition camera (frame rate ≥30 fps) for no less than 1 hour. The relevant behavioral parameters are counted by image analysis software. The respiratory rate is counted by manually counting the number of times the gill cover opens and closes. Each count lasts for 1 minute and is repeated 3 times with a 5-minute interval. The average value is taken.

[0066] (1) Characteristics of activity pattern change: The high-definition camera was used to continuously record for 1 hour, and the resting time, number of movements per unit time and number of times the appendages were upright were statistically analyzed using image analysis software.

[0067] Activity frequency increase rate = (Number of moves during the detection period - Number of moves during the resting period) / Number of moves during the resting period × 100%; The resting period is 2-4 days before the mud crab molts. During this time, the mud crab reduces or stops feeding, does not move, and rests quietly in a secluded corner. The resting period usually lasts 1-2 days. The number of times the mud crab moves during this period is used as the number of times it moves during the resting period.

[0068] Judgment criteria: After resting for 24-48 hours, if the activity frequency increases by 300% or more, and the appendages are upright ≥5 times per hour and each time lasts ≥15 seconds, showing that the walking legs or chelipeds support the body vertically and the head and thorax are raised off the ground, then the characteristics are met.

[0069] (2) Tissue softening characteristics of specific parts: The hardness of the abdominal carapace below the longest serration on both sides of the cephalothorax of the mud crab was measured. The hardness of the abdominal carapace was measured using a Shore hardness tester (Type A). The instrument was calibrated before measurement. During measurement, the probe was perpendicular to the surface of the abdominal carapace and a pressure of 1N was applied. Each measurement site was measured 3 times, and the measurement was performed 3 times a day. The average value was compared with the average hardness of the same part of the individual in the non-molting period to calculate the hardness reduction rate.

[0070] Hardness reduction rate = (Penal plate hardness during non-molting period - Penal plate hardness during testing period) / Penal plate hardness during non-molting period × 100%; Judgment criteria: The characteristic is met when the rate of decrease in hardness is 20% or more lower than that of individuals in the non-molting period.

[0071] (3) Increased respiratory rate: Manually count the number of times the gill cover opens and closes, each count lasts for 1 minute, repeat 3 times with a 5-minute interval, and take the average value to calculate the respiratory rate.

[0072] Judgment criteria: A respiratory rate of 13-16 breaths / minute, which is 30% or more higher than the normal level, is considered to meet the criteria.

[0073] (4) Wiping behavior characteristics: The camera continuously recorded for 1 hour, and the number of times the chelicerae scraped the joint between the plastron and carapace and the duration of each action were counted by image analysis software. The total daily cleaning behavior time was accumulated.

[0074] Judgment criteria: ≥20 actions per hour, each action lasting ≥10 seconds, and a total daily cleaning time of ≥5 minutes are required to meet the criteria.

[0075] Example 3 This embodiment provides experimental verification of pre-molting identification in mud crabs. It verifies the accuracy of identification using five quantitative characteristics from Example 2 (enhanced feeding behavior, changes in appendage joints, cephalothorax edge characteristics, changes in abdominal setae and color, and changes in abdominal morphology) during the pre-molting period (12-15 days before molting) in mud crabs. The results are compared with the control group using relevant literature (Hou Yiling, Cheng Wenzhi, Wei Yiming, et al. Research progress on the identification technology of molting cycle stages in shrimp and crabs [J]. Fisheries Science). ,2024,43(02):319-332.DOI:10.16378 / j.cnki.1003-1111.22058;Xuan Fujun, Jiang Senhao, Bian Xunguang, et al. Reproductive molting and mating behavior of blue crab under indoor culture conditions[J]. Zoological Journal,2014,49(04):579-586.DOI:10.13859 / j.cjz.201404015.;Kennedy, VS,&Cronin, LE (2007).The blue crab: Callinectes sapidus.) Comparison of indicators.

[0076] In this embodiment, the experimental bait was divided into two categories: high-protein live bait, which consisted of small fish (sardines), shrimp (whiteleg shrimp), and shellfish (razor clams) with a protein content of ≥40%, was chopped and mixed with low-protein plant bait (corn, soybean cake, etc.) in a fixed ratio (1:1) and fed to the crabs. The crabs' preference for high-protein live bait and low-protein plant bait was evaluated by observing their feeding behavior and selection preferences for the two types of ingredients in the mixed bait.

[0077] The aquaculture water is filtered seawater with a salinity of 12-18%, pre-aerated for 24 hours, and the temperature is stabilized at 24-26℃ with a dissolved oxygen content ≥5.0mg / L. The recirculating aquaculture tanks are single tanks with dimensions of 60cm×40cm×30cm, equipped with simple hiding places, and use a recirculating aquaculture system (RAS), model AquacareSystemsRAS-2000.

[0078] The specific experimental steps are as follows: Select the mud crab that is used for burrowing ( Scylla paramamosain ), Sawtooth blue crab ( Scylla serrata Two hundred healthy individuals from each species were selected, with a body width of 3-5 cm and a weight of 20-30 g, and were in the stable period (non-molting period) 7-15 days after molting. The two species of mud crabs were randomly divided into an experimental group and a control group, with 100 crabs in each group, and were raised separately in breeding tanks of the same size.

[0079] Experimental group: The five quantitative characteristics specified in Example 2 for the early molting stage were used as detection indicators to determine whether the mud crab had entered the early molting warning stage.

[0080] Control group: Early molting characteristics obtained from the above-mentioned relevant literature were used as detection indicators, including body surface color, color of the swimming foot joints, presence or absence of molting sutures, and degree of curvature of the xiphoid process.

[0081] The specific operating procedure is as follows.

[0082] Experimental group operating procedures: The feeding behavior of mud crabs was recorded at fixed times every day (8:00, 14:00, 22:00), and the daily feed intake and preference for high-protein live bait were statistically analyzed.

[0083] Five quantitative characteristics of the pre-molting stage in Example 2 were measured daily using standardized instruments for 15 consecutive days. The accuracy and duration of each characteristic were recorded. When the mud crab met at least three of the five characteristics for at least three consecutive days, it was marked as a predicted entry into the pre-molting stage. The actual molting time was tracked and recorded to verify the accuracy of the prediction.

[0084] Control group procedure: At fixed times each day (8:00 AM and 4:00 PM), the following indicators were recorded using corresponding instruments: whether the skin color lightened, whether black marks appeared on the soles of the feet during swimming, whether peeling sutures appeared, and whether the xiphoid process showed slight curvature. Details are as follows: Changes in body surface color were measured using standard colorimetric cards, just like in the experimental group.

[0085] The pigment deposition in the soles of swimmers' feet was detected using dermoscopy. The specific procedure was as follows: high-resolution imaging of the swimmer's footprint area was performed using a dermoscope, followed by image analysis software to process and quantitatively evaluate the acquired images, focusing on the structural characteristics such as the uniformity of epidermal pigment deposition distribution and deposition depth.

[0086] The observation of molting sutures was performed using a magnifying glass. The specific procedure was as follows: the crab was fixed in a dissecting tray and a magnifying glass (10× magnification) was used to systematically examine the dorsal region of the cephalothorax. The focus was on whether there were any tiny cracks or weak areas in the shell caused by water absorption at the junction of the carapace and the plastron (i.e., the posterior edge of the plastron).

[0087] The degree of curvature of the scimitar was detected using image analysis. By taking a photo of the scimitar, the curvature parameters were measured using image processing software (such as ImageJ). Specifically, the crab's head was placed flat, and a clear side view of the scimitar was taken with a mobile phone or camera. The photo was imported into ImageJ software, and the contrast was adjusted to make the outline of the scimitar clear. The line segment tool in ImageJ software was used to measure the chord length (L) and arc height (h); the angle tool in ImageJ software was used to measure the curvature angle (θ). The measurement was repeated 3 times, and the average value was taken.

[0088] When ≥3 traditional characteristics change, it is marked as a predicted entry into the pre-molting stage. The actual molting time is tracked and recorded to verify the accuracy of the prediction.

[0089] The characteristics of appendage joint changes, carapace edge features, abdominal umbilical setae length and color changes, and abdominal morphological changes in the experimental group were all based on the characteristics detected in non-molting mud crabs, as well as those in the control group.

[0090] The experimental results are shown in Tables 2-4 below. Tables 2 and 3 show that the experimental group achieved a 97.9% accuracy rate in identifying the pre-molting stage with a false positive rate of only 2.1%, significantly better than the control group's 65.9% accuracy rate and 34.1% false positive rate. This indicates that the five quantitative features for predicting the molting stage of mud crabs can accurately capture the physiological changes during this period. Table 4 shows that the average satisfaction rate of each feature in the experimental group among correctly identified individuals was between 93.6% and 100%. Among them, the satisfaction rate of the appendage joint changes and the cephalothorax edge features both reached 100%, indicating that these two features are the core signals for the pre-molting stage and have strong stability. The traditional indicators in the control group were greatly affected by subjective observation. For example, there were individual differences in judging the body surface color and the color of the swimming foot segments. Furthermore, features such as the molting suture and the curvature of the rostrum appeared later, resulting in a lower accuracy rate and failing to effectively achieve pre-molting warning.

[0091] There was no significant difference in the identification accuracy between the two types of mud crabs in the experimental group, indicating that the method of the present invention is applicable to the identification of different species of mud crabs in the pre-molting stage and has strong versatility. The number of correctly identified individuals in the experimental group was 94 for *Scylla serrata* and 93 for *Scylla serrulata*, providing a sufficient and qualified source of experimental subjects for subsequent studies.

[0092] Table 2. Identification data of the experimental group in the early molting stage

[0093] Table 3. Identification data of the control group in the early molting stage

[0094] Table 4. Statistics on the Satisfaction of Various Characteristics in the Experimental Group

[0095] Example 4 This embodiment provides experimental verification of the identification of the molting period, verifying the accuracy of the four quantitative characteristics of the molting period in Example 2 (activity pattern change characteristics, tissue softening characteristics in specific areas, increased respiratory rate characteristics, and wiping behavior characteristics) for the identification of the molting period (within 1-2 days) of mud crabs. The specific experimental steps are as follows, by comparing with the relevant literature indicators of the control group.

[0096] The experimental breeding conditions and feed used in this embodiment are the same as in embodiment 3, and the ceramic pipes and tiles are placed in the corners of the recirculating aquaculture tank.

[0097] In Example 3, mud crabs that were correctly predicted to be in the pre-molting stage were selected. After molting, they were again predicted to be in the pre-molting stage and used in this example. Seventy mud crabs (Scylla serrata) and seventy mud crabs (Scylla serratedis) were selected, all healthy individuals, with a body width of 5-7 cm and a weight of 40-60 g, clearly indicating they had entered the pre-molting stage (12-15 days before molting). Seventy mud crabs were randomly selected from each of the correctly predicted individuals to form experimental and control groups: 35 mud crabs in the experimental group and 35 in the control group; and 35 mud crabs in the experimental group and 35 in the control group. These were raised separately in identical tanks.

[0098] Experimental group: Four quantitative characteristics of the molting stage in Example 2 were used as detection indicators to determine whether the mud crabs had entered the molting stage. Each group consisted of 35 crabs.

[0099] Control group: The characteristics of imminent molting obtained from relevant literature in Example 3 were used as detection indicators, including reduced or stopped food intake, reduced or stopped activity, reduced aggression, and overall decrease in shell hardness. There were 35 animals in each group.

[0100] The specific experimental steps are as follows: Specific operating procedures for the experimental group: Starting on the 10th day of the pre-molting phase, the behavior, carapace hardness, and respiratory rate of the mud crabs were continuously monitored four times daily (8:00, 12:00, 16:00, and 22:00). High-definition cameras were used to continuously film the crabs' behavior for at least one hour each time. Image analysis software was used to statistically analyze activity frequency, number of times appendages were erected, and number of wiping movements. The hardness of the plastron region below the longest serrations on both sides of the cephalothorax was measured using a Shore A hardness tester, with three measurements taken daily and the average value recorded. The number of times the gill covers opened and closed was manually counted, with each count lasting one minute, repeated three times at 5-minute intervals, and the average value was used to calculate the respiratory rate.

[0101] When a mud crab simultaneously meets all four characteristics of the molting period in Example 2, it is marked as molting within 1-2 days. The actual molting time is tracked and recorded to verify the accuracy of the prediction.

[0102] Specific operating procedures for the control group: Starting on the 10th day of the pre-molting phase, the traditional pre-molting characteristics of mud crabs were monitored four times daily (8:00, 12:00, 16:00, and 22:00). Changes in food intake were observed and recorded. Record daily food intake; a reduction of ≥50% from normal levels or cessation of food intake is considered a satisfactory characteristic. Activity level: Observe the movement frequency of mud crabs. A decrease of ≥80% or cessation of activity compared to normal levels within 1-2 days is considered a satisfactory characteristic. Aggression: The crab is deemed to have a positive characteristic if it does not retaliate when its claws are gently touched with a small glass rod. Carapace hardness: Pressing the central area of ​​the carapace with your finger, it should feel noticeably softer, indicating that the test is satisfactory.

[0103] When a mud crab simultaneously meets all ≥4 characteristics of the control group, it is marked as being in the molting period, and molting is predicted to occur within 1-2 days. The actual molting time is tracked and recorded to verify the accuracy of the prediction.

[0104] The baseline for the softening characteristics of specific tissues in the experimental group and the characteristics of the control group were both based on the characteristics measured by non-molting mud crabs.

[0105] The experimental results are shown in Tables 5-7 below. Tables 5 and 6 show that the experimental group achieved an accuracy rate of 97.1% in identifying the molting period, with a misjudgment rate of only 2.9%, which is significantly higher than the control group's accuracy rate of 70.9% and misjudgment rate of 29.1%. This indicates that the four quantitative features of the present invention can accurately capture the behavioral and physiological signals of mud crabs nearing molting. Table 7 shows that the average satisfaction rate of various features in actual molting individuals in the experimental group was between 91.2% and 97.1%. Among them, the activity pattern change feature and the increased respiratory rate had the highest satisfaction rate (both 97.1%), which are key indicators for judging the molting period, with clear signals that are not easily interfered with. The traditional indicators in the control group are mostly qualitative descriptions, such as obvious softening of the shell and reduced activity, which are subjective. Moreover, some features (such as cessation of activity) may be caused by non-molting factors such as environmental stress, leading to a higher misjudgment rate.

[0106] The difference in identification accuracy between the two types of mud crabs in the experimental group was minimal, further verifying the versatility of the method of the present invention, applicable to accurate prediction of the molting period of different mud crab varieties. The experimental subjects were all from the correctly identified individuals in Example 3, ensuring the consistency and effectiveness of the experimental samples and avoiding interference from differences in the basic state of the samples, thus making the verification conclusions more reliable.

[0107] Table 5. Identification data of the experimental group near molting stage

[0108] Table 6. Identification data of the control group near molting period

[0109] Table 7. Statistics on the Satisfaction of Various Characteristics in the Experimental Group

[0110] Example 5 This embodiment provides an accuracy verification of the molting prediction method for mud crabs. It verifies the prediction accuracy of the prediction method in Example 3 in two stages: the pre-molting stage (12-15 days before molting) and the near-molting stage (1-2 days) in Example 4. It compares the misjudgment rate of the feature judgment method in relevant literature in Example 3. The breeding environment and feed are the same as in Example 3. The specific experimental steps are as follows.

[0111] Select the mud crab that is used for burrowing ( Scylla paramamosain ) and serrated blue crab ( Scylla serrata 200 individuals were selected from each group, with a body width ranging from 8 to 10 cm and a weight ranging from 150 to 200 g. The difference in body width was ≤5% and the difference in weight was ≤10%. All individuals were healthy and in a stable period of 7 to 20 days after molting. The experimental period was 60 days, covering the complete molting cycle of mud crabs.

[0112] Experimental group: The multi-index fusion prediction method based on behavioral and morphological features, as described in Example 2 of this invention, was used to identify and predict the molting stage of mud crabs. The specific operation is as follows: On day 1 of the experiment, 200 mud crabs were numbered, and basic data (appendage joint diameter, carapace hardness, etc.) were measured as reference values ​​for normal condition. High-definition cameras were used to continuously record images for one hour each day at 9:00, 15:00, and 21:00. Image analysis software was used to statistically analyze activity frequency, number of times appendages were erected, and number of wiping movements. Respiratory frequency was manually counted for one minute each time, repeated three times with a 5-minute interval, and the average value was taken. Every three days, appendage joint diameter and color value, width of the cephalothorax edge protrusion, and vertical distance between the plastron and cephalothorax were measured, and feeding information (daily food intake, food preference) was recorded. When a mud crab met at least three of the five pre-molting characteristics for ≥3 days, it was considered to be in the pre-molting stage; when it met all four characteristics of impending molting, it was considered to be in the impending molting stage. The warning time and actual molting time were recorded.

[0113] Control group: Combining the determination methods of the control group in Examples 3 and 4, technicians with more than 5 years of breeding experience made molting predictions based on traditional experience (relying on subjective observations such as body surface color and activity level), and recorded the predicted molting time.

[0114] The experimental results are shown in Table 8 below. The experimental group achieved a comprehensive prediction accuracy of 97.5%, with a pre-molting early warning accuracy of 97.0% and a near-molting confirmation accuracy of 98.0%, and a misjudgment rate of only 2.5%. In contrast, the control group achieved a comprehensive accuracy of only 58.5% and a misjudgment rate of 41.5%. This demonstrates that the present invention, through multi-indicator quantitative fusion judgment, effectively avoids the influence of environmental fluctuations and individual differences, improving prediction accuracy by 66.7% (an absolute improvement of 39.0%) compared to traditional methods. It can accurately pinpoint the key time nodes for molting in mud crabs, providing a reliable basis for subsequent management.

[0115] Table 8. Experimental data verifying the accuracy of the molting prediction method for mud crabs.

[0116] Example 6 This embodiment verifies the effectiveness of management measures following an early warning system for molting, specifically examining the impact of water quality regulation, feed optimization, and the deployment of concealed structures on the molting success rate and new shell development quality of mud crabs after such an early warning. The specific experimental steps are as follows.

[0117] Two hundred mud crabs of the same size as in Example 3 were selected and determined to be in the early molting stage (12-15 days before molting) by the method in Example 2 of this invention. The experimental period was 15 days, from the early molting warning to the completion of molting.

[0118] Experimental group: The aquaculture management was carried out using the supporting control measures following the early warning of the molting stage of this invention, as detailed below: (1) The water body has a stable pH value of 7.8-8.2, a temperature of 24-26℃, and a salinity of 12-18%.

[0119] (2) Install concealed structures such as ceramic pipes, tiles and wire mesh cages to achieve an environmental shading rate of 60-70%.

[0120] (3) Gradually increase the calcium ion concentration in the water to 400-500 mg / kg and maintain dissolved oxygen at 5.5-6.0 mg / L.

[0121] (4) Feed 3 times a day, with each feeding amount being 3-4% of the crab's body weight, using high-protein fresh bait.

[0122] (5) Maintain a light intensity of 500-1000 lx and a photocycle of 12 hours of light and 12 hours of darkness per day; control ammonia nitrogen ≤0.1 mg / L and nitrite ≤0.05 mg / L.

[0123] Control group: Traditional and conventional breeding and management methods were used without any targeted adjustments, as detailed below: (1) Natural water quality environment (pH, calcium ion concentration, etc. are not adjusted) and no concealed structures are installed.

[0124] (2) Feed twice a day, with each feeding amount being 2-3% of the body weight. Use conventional mixed feed (a mixture of high-protein feed (fish, clams) and low-protein feed (corn, soybean cake)).

[0125] (3) No specific light, temperature and salinity control measures.

[0126] The experimental results are shown in Table 9 below. The molting success rate of the experimental group reached 95.0% (95 animals successfully molted), which is 27.0 percentage points higher than that of the control group (68.0%, 68 animals successfully molted). The growth rate of new shell hardness was 42.3%, which is significantly higher than that of the control group (28.5%). Specifically, the average hardness of the plastron in the experimental group was 28.6 Shore A (HA) on the first day after molting, which increased to 40.7 Shore A (HA) on the tenth day, while the hardness in the control group only increased from 27.9 Shore A to 35.9 Shore A. This indicates that the precise increase in calcium ion concentration and nutritional support in the targeted regulation provided sufficient raw materials for the synthesis of calcium carbonate in the new shell, effectively promoting the hardening and development of the new shell.

[0127] The experimental group achieved a feed utilization rate of 76.8% (total weight gain of 3126g, total feed intake of 4070g), a 24.3 percentage point increase compared to the control group's 52.5% (total weight gain of 1848g, total feed intake of 3520g). This improvement was attributed to the use of high-protein live bait (fish, shrimp, and shellfish) and a precise feeding regimen of three times a day, with each feeding representing 3-4% of the crab's body weight. This approach matched the high metabolic demands of mud crabs during the pre-molting period while minimizing feed waste. Furthermore, the experimental group achieved a 97.9% survival rate (93 crabs survived) 10 days after molting, significantly higher than the control group's 82.4% (56 crabs survived). The stable calcium ion concentration in the water (92.0%) was also significantly better than the control group's 65.0%, demonstrating the crucial role of a stable water environment and concealed structural features in reducing stress and improving the health of mud crabs.

[0128] The pre-molting stage is crucial for mud crabs to accumulate nutrients and build a new shell. Environmental parameters and aquaculture management measures must be precisely matched to their physiological needs: a water pH of 7.8-8.2 is suitable for mud crab growth and new shell synthesis; excessive acidity or alkalinity will affect calcium absorption and enzyme activity; a temperature of 24-26℃ and a salinity of 12-18% can maintain a stable metabolic rate for mud crabs, avoiding stress responses caused by temperature or salinity fluctuations; calcium ions are the core component of calcium carbonate in the new shell, and a concentration of 400-500 mg / kg can meet the calcium requirements for new shell synthesis; dissolved oxygen of 5.5-6.0 mg / L can ensure oxygen supply during periods of high feeding; setting up concealed structures and controlling the shading rate to 60-70% can reduce mutual interference and cannibalism among mud crabs, thus reducing stress responses; feeding three times a day with 3-4% high-protein feed per feeding can match the high feeding needs of mud crabs during the pre-molting stage, ensuring sufficient protein and energy intake; 500-1000 The 12L:12D light intensity and photoperiod align with the natural growth rhythm of mud crabs, promoting stable physiological metabolism. Maintaining low concentrations of ammonia nitrogen and nitrite avoids damage to the hepatopancreas and gills, ensuring the crabs' health. These combined regulatory measures create a suitable environment for the early molting stage of mud crabs. This approach aims to create the optimal environment for growth and nutrient accumulation, specifically addressing the crabs' core needs for nutrition, water quality, and environment during this phase. It promotes normal new shell development, strengthens the crabs' constitution, and reduces problems such as abnormal growth and molting difficulties caused by unsuitable environments or insufficient nutrition, laying a solid foundation for successful molting later.

[0129] Table 9. Data on the effectiveness of management measures after early warning of molting.

[0130] Note: 1. The hardness of the plastron was measured using a Shore A hardness tester. The plastron region below the longest serration on both sides of the cephalothorax was measured for each crab, and the average value was taken for each measurement; 2. The calcium ion concentration in the water was measured twice a day at 9:00 AM and 5:00 PM using a calcium ion analyzer, and the average value for that day was taken; 3. Total weight gain = total weight of crabs at the end of the experiment - total weight of crabs at the beginning of the experiment; 4. The total amount of feed was the sum of the dry weight of the feed actually fed during the experiment.

[0131] Example 7 This embodiment provides an effectiveness verification of emergency intervention measures near the molting stage, verifying the impact of measures such as increasing temperature, reducing light exposure, and increasing dissolved oxygen during the molting stage on the molting process and molting mortality. The specific experimental steps are as follows.

[0132] Two hundred mud crabs were selected and determined to be in the molting stage (expected to molt within 1-2 days) according to the method of Example 2 of this invention. The size was the same as in Example 4. The experimental period was 5 days, and the molting time and molting mortality rate were recorded.

[0133] Experimental group: The emergency control measures for the period nearing molting according to the present invention were adopted, as follows: (1) The water temperature is increased by 2-3℃ at a heating rate of 0.3℃ per hour, and finally stabilized at 26-28℃.

[0134] (2) Cover with a blackout cloth to reduce the light intensity to 100-200 lx and adjust the light cycle to 10 hours of light and 14 hours of darkness.

[0135] (3) Add 0.8 g / m³ of water to the water body according to the ratio of usage to water volume. 3 Vitamin C at 0.25g / m 3 EDTA-2Na.

[0136] (4) Adjust the dissolved oxygen content in the water to 6.5-7.5 mg / L and stop feeding.

[0137] Control group: Traditional breeding management was adopted without any emergency intervention measures, as detailed below: (1) Maintain the original water temperature (24-26℃) and light intensity (natural light, about 5000 lx).

[0138] (2) Do not add vitamin C and EDTA-2Na, maintain dissolved oxygen at 5.0-5.5 mg / L, and feed normally.

[0139] The experimental results are shown in Table 10 below. The average molting time in the experimental group was 45.2 minutes, which was 33.4 minutes shorter than the 78.6 minutes in the control group. The molting mortality rate was only 4.0% (4 deaths), far lower than the 32.0% (32 deaths) in the control group. The incidence of stress response was 8.0% (8 animals), which was 37.0 percentage points lower than the 45.0% (45 animals) in the control group. The molting success rate reached 96.0%, significantly better than the 68.0% in the control group. At the same time, the hardening rate of the new shell in the experimental group was 2.3 hours / hardness unit, which was significantly faster than the 3.8 hours / hardness unit in the control group. This indicates that the emergency intervention measures not only promoted successful molting but also provided favorable conditions for the development of the new shell.

[0140] During the molting period, mud crabs are physiologically sensitive and have weak adaptability. Various control measures are precisely implemented around "reducing stress, promoting molting, and ensuring survival": the temperature is increased to 26-28℃ at a rate of 0.3℃ per hour to gradually increase the metabolic rate and molting hormone secretion, avoiding rapid temperature increase stress; the light intensity is reduced to 100-200 lx with a 10L:14D photoperiod to match their photophobic behavior and reduce external stimulation; vitamin C and EDTA-2Na are added to enhance stress resistance and chelate heavy metal ions, respectively, to protect the fragile epidermis during molting; dissolved oxygen is increased to 6.5-7.5 mg / L to meet the surge in oxygen demand during molting and avoid molting interruption due to hypoxia; feeding is stopped to reduce the burden on the digestive tract and pollution from uneaten feed, ensuring clean water quality. These measures create a low-stress, highly adaptable molting environment, effectively reducing the risks of stress response, hypoxia, and water pollution, shortening molting time, reducing problems such as molting difficulties, limb loss, and death, and significantly improving the molting success rate and post-molting survival rate. This has important practical guiding significance for the large-scale farming of mud crabs.

[0141] Table 10. Data on the effectiveness of emergency intervention measures near the molting period.

[0142] Note: 1. Molting time and new shell hardness data were monitored and recorded 24 hours a day using a Huawei full-color 4K high-definition camera system. Hardness measurements were taken from the central area of ​​the plastron, with three measurements taken each time and the average value taken. 2. Stress response was determined by a combination of video playback and on-site observation, focusing on recording abnormal struggles, appendage breakage, and incomplete shedding of the old shell during molting. 3. The time to complete hardening was started from the completion of molting (when the chelipeds were completely removed from the old shell) until the difference between two consecutive hardness measurements (12 hours apart) was less than 5%.

[0143] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for predicting the molting period of mud crabs, characterized in that, The molting period includes the pre-molting stage and the period immediately before molting; The pre-molting period refers to 12-15 days before the mud crab molts, and the imminent molting period refers to 1-2 days before the mud crab molts. The method includes the following steps: acquiring behavioral and morphological data of mud crabs, and predicting the pre-molting and near-molting periods of mud crabs; The behavioral data includes the feeding behavior, activity frequency, respiratory rate, and wiping behavior of the mud crabs; The morphological data includes the appendage joint features, cephalothorax edge features, length of abdominal setae, abdominal setae color, abdominal morphological variations, and carapace hardness of the mud crab. Among them, the activity frequency, number of times appendages were erected, and wiping behavior of mud crabs were obtained through image analysis software. The length of the abdominal setae of the mud crab was obtained by acquiring high-definition images of the setae region on the outer edge of the abdominal navel using a high-precision image measuring instrument. Among them, when the mud crab meets at least 3 of the following conditions (1)-(5), it is predicted that the mud crab has entered the pre-molting stage: (1) The daily food intake of mud crabs is 20% or more higher than the normal baseline level, and they prioritize protein intake; the normal baseline level is the average daily food intake of mud crabs 7-20 days after molting; the protein includes at least one of fish, shrimp and shellfish. (2) The diameter of the limb joint increases by 3% or more compared to the non-molting period; at the same time, the epidermal color value at the joint decreases by 10% or more compared to the normal area; the normal area is the area adjacent to the joint. (3) The posterior and lateral margins of the cephalothorax of the blue crab form continuous raised lines, and the width of the raised lines is greater than or equal to 0.5 mm; (4) The length of the bristles on the outer edge of the abdomen of the mud crab increases by 20% or more compared with the non-molting period, when the color value of the abdomen navel L is ≥80 and the b value is 20-30; the color value of the abdomen navel L and b are obtained by acquiring the image of the abdomen navel area under a standard light source using the CIELab color space. (5) The vertical distance between the central part of the plastron and the posterior edge of the cephalothorax increases by 5% or more compared to the non-molting period when the body width is similar; The non-molting period is 7-15 days after molting; When a mud crab enters the pre-molting stage, if it meets all of the following characteristics (1)-(4), then it is predicted that the mud crab is entering the near-molting stage: (1) After the mud crab has been lying still for 24-48 hours, the number of times it moves per hour is 300% or more higher than during the lying-still period; and the crab appears in an upright position with its appendages at least 5 times per hour, each time lasting at least 15 seconds. (2) The hardness of the abdominal carapace below the longest serrations on both sides of the cephalothorax of the mud crab is reduced by 20% or more compared with the non-molting period; (3) The number of times the gill covers open and close is counted. The breathing rate of the blue crab reaches 13-16 times per minute. (4) The number of times the mud crab scrapes the seam between the abdominal carapace and the carapace with its claws shall not be less than 20 times per hour, each time lasting not less than 10 seconds, accompanied by the cleaning action of the walking legs on the surface of the body, and the total daily wiping time shall not be less than 5 minutes.

2. The method according to claim 1, characterized in that, The changes in abdominal morphology include variations in the vertical distance between the central part of the plastron and the posterior edge of the cephalothorax.

3. The method according to claim 1, characterized in that, The hardness of the carapace includes the hardness of the abdominal plate area below the longest serrations on both sides of the cephalothorax of the blue crab.

4. The method according to claim 1, characterized in that, The mud crabs mentioned include mud crabs such as mud crab, mud crab, mud crab, mud crab or hybrids thereof.

5. A method for farming mud crabs, characterized in that, The method includes the following steps: after predicting the pre-molting stage using the method for predicting the molting period of mud crabs according to any one of claims 1-4, the following steps (1)-(5) are performed: (1) Increase the ambient shading rate to 60-70%; (2) Increase the calcium ion concentration in the water to 400-500 mg / kg and maintain dissolved oxygen at 5.5-6.0 mg / L; (3) Increase the feeding frequency to 3-5 times, with a single feeding amount of 3-4% of the body weight of the mud crab; the feeding includes feeding with feed from at least one source of fish, shrimp and shellfish; (4) Adjust the light intensity to 500-1000 lx; (5) Control the ammonia nitrogen in the water body to ≤0.1 mg / L and the nitrite to ≤0.05 mg / L.

6. A method for farming mud crabs, characterized in that, The method includes the following steps: after predicting the approaching molting period using the method for predicting the molting period of mud crabs according to any one of claims 1-4, perform any one or more of the following steps (1)-(6): (1) Raise the water temperature to 26-28℃; (2) Adjust the light intensity to 100-200 lx; (3) Adjust the photoperiod to 8-10 hours of light and 14-16 hours of darkness; (4) Add 0.5-1 g / m³ to the water body according to the ratio of usage to water volume. 3 Vitamin C at 0.1-0.5 g / m 3 EDTA-2Na; (5) Adjust the dissolved oxygen in the water to 6.5-7.5 mg / L; (6) Stop feeding.

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