Method for evaluating acute thermal stress in flatfish and use thereof

Abstract

The present application belongs to the technical field of seawater fish breeding and breeding, and particularly relates to a method for evaluating temperature acute stress of flatfish and application. The flatfish is put into a breeding area, an experimental water flow velocity is set, the water body is gradually warmed or cooled, when the flatfish cannot lie flat on the water bottom and is carried into a detection position by the water flow, the temperature or time corresponding to the moment is recorded, that is, the tolerance temperature or tolerance time of the flatfish; wherein the detection position is close to the water outflow direction side of the breeding area. The evaluation method of the present application can determine whether the fish can lie flat on the water bottom through the water flow, and takes being carried to the detection position as the standard, which can avoid the stress reaction of the fish caused by artificial stirring, and is more objective and accurate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of marine fish culture and breeding, and specifically relates to a method for accurately and efficiently and simply evaluating the temperature stress tolerance limit of flatfish such as flatfish and other benthic fish on the seabed and application thereof. BACKGROUND

[0002] Flatfish mainly refers to fish of the order Pleuronectiformes, also known as flatfish. Most of them have high economic value. For example, Paralichthys olivaceus, Scophthalmus maximus and Cynoglossus semilaevis are major marine aquaculture varieties in northern China. They have also promoted the development of related industries in various coastal provinces in China. The culture of these fish directly led to the rise of industrialized aquaculture in China.

[0003] With global climate change, the high temperature in summer is higher, the low temperature in winter is lower, and the duration is longer. This directly causes adverse effects on fish in net cage culture and pond culture. In industrialized culture, corresponding cooling or warming is also needed, which consumes a large amount of energy and greatly increases the cost of culture, which is not conducive to energy conservation and environmental protection and sustainable development. Higher or lower temperature not only retards growth, but also causes susceptibility to pathogens and diseases, which brings the risk of disease outbreak, and leads to the limitation of the range and scale of culture. In view of this situation, it is urgent to breed new temperature-tolerant varieties in production to improve the survival rate in summer and overwintering culture, reduce the energy consumption of temperature control, improve the culture benefit, and expand the culture range.

[0004] To breed new temperature-tolerant varieties, it is necessary to first screen temperature-tolerant families or individuals, and then construct temperature-tolerant inbred lines. Temperature-tolerant families or individuals usually need to be evaluated for temperature tolerance through temperature stress experiments, and then screened.

[0005] The temperature tolerance of general swimming fish can be judged by sinking to the bottom of the water or lying on the back or even turning over when swimming, which can be observed directly by naked eyes. However, for fish of the order Pleuronectiformes, Rajiformes and Sebastidae, such as Pseudosciaena crocea and Liachirus asper, the fish often lie on the bottom of the water when living, and the body is often rigid when the fish is unbalanced or dead, and the fish still maintains the state of lying on the bottom of the water as when living, so it is difficult to judge whether the fish is unbalanced or dead, and the method for judging the temperature tolerance of the fish is often to judge the state of each fish by moving the fish, but moving the fish will also cause stress to the fish, and affect the accuracy of the judgment of the temperature tolerance. Moreover, the judgment of the unbalance of the fish is artificial, and the subjective factor has a great influence, and the efficiency is also low, and a long time of tracking observation is needed, so the method of the prior art has poor effect. Specifically, when the flatfishes and other fish that lie on the bottom of the water reach the temperature tolerance limit, the fish will be unbalanced or dead, and the body is rigid, but it is difficult to directly judge by naked eyes, and it is often necessary to artificially move each fish to observe whether the fish is unbalanced, which causes great stimulation to the fish, and even causes stress, and affects the result of the judgment of the temperature tolerance. At the same time, when the fish is artificially moved, the fish is often turned over to the eyeless side, and whether the fish can turn back to the original state is judged to speculate whether the fish is unbalanced or dead. Many fish can struggle in a half-unbalanced or half-stressed state, and can turn back to the original state after a period of time, but the turning back time of different individuals is different, and artificial judgment is needed, the subjective factor has a great influence, and a long time is needed. These factors have an adverse effect on the precise selection of temperature tolerance phenotypes for breeding application, and thus it is urgent to develop a precise and effective method for evaluating the temperature stress of flatfishes. It is found that, under the artificial breeding conditions, the flatfishes usually tightly adhere the eyeless side and the odd fins (dorsal fin, anal fin and tail fin) to the bottom of the breeding pool to reduce the impact of the water flow and prevent being washed away by the water flow. After being anesthetized, the individuals cannot tightly adhere to the bottom of the breeding pool, and the small water flow can also wash them away. According to the theory of fluid mechanics and the existing reports, with the increase of the water flow speed, the impact force of the water flow on the fish increases rapidly in an exponential manner, and is proportional to the square of the flow speed. When the water flow speed increases, the fish needs to consume certain energy to resist the impact of the water flow, and changes the body posture, and often shows muscle tension and slight bulging of the body back. Too high flow speed will cause stress to the fish, and the metabolism and stress indicators change, and the oxygen consumption rate increases. Therefore, the present application is aimed at the problems in the temperature stress research and application of flatfishes and other flatfishes, and for the first time, the water flow with a suitable flow speed is used to screen the individuals reaching the temperature tolerance limit. The application of the device and the method not only avoids the stress caused by moving the fish, but also is efficient and simple, and is more objective. The related content of the present application has not been reported in the research and application of fish temperature tolerance, and can provide support for the breeding research of the temperature tolerance traits of flatfishes and other fish that lie on the bottom of the water. SUMMARY

[0006] The present application aims to provide a device and method for evaluating the temperature stress of flatfishes.

[0007] To achieve the above object, the technical scheme of the present application is as follows:

[0008] A method for evaluating acute temperature stress of flatfish, the flatfish is put into a culture area, an experimental water flow rate is set, the water body is gradually warmed or cooled, when the flatfish cannot lie flat on the water bottom and is carried into a detection position by the water flow, the temperature or time corresponding to the moment is recorded, that is, the tolerance temperature or tolerance time of the flatfish; wherein the detection position is close to the water flow out direction side of the culture area.

[0009] Further, the flatfish is put into the culture area, and after temporary culture for 24 hours, the experimental water flow rate is set, the temperature is increased or decreased at a rate of 0.5℃ / h-1.0℃ / h, until the flatfish cannot lie flat on the water bottom; then the temperature is continuously increased or decreased at a rate of 0.1℃ / h-0.2℃ / h, until all the flatfish cannot lie flat on the water bottom and are carried into the detection position by the water flow;

[0010] When the flatfish individual is carried into the detection position by the water flow, it is fished out in time, and the temperature or time corresponding to the moment is recorded, that is, the tolerance temperature or tolerance time of the flatfish individual.

[0011] The experimental water flow rate determination method is: minimum water flow rate ≤ experimental water flow rate ≤ maximum water flow rate;

[0012] The minimum water flow rate is: a plurality of flatfish are put into the culture area after being anesthetized, the flow rate is gradually increased at a rate of 0.1m / s-0.2m / s per hour from 0m / s, until all the flatfish are carried into the detection position by the water flow, and the flow rate is recorded as the minimum water flow rate;

[0013] The maximum water flow rate is: a plurality of flatfish are put into the culture area, and after 24 hours, the average breathing frequency of the flatfish at rest is recorded; then, the flow rate is gradually increased at a rate of 0.1m / s-0.2m / s per hour from the minimum flow rate, the average breathing frequency of the flatfish is recorded, until the average breathing frequency is increased by 30%-50% compared with the average breathing frequency at rest, and the flow rate is recorded as the maximum water flow rate;

[0014] The average breathing frequency is the average number of breathing times calculated continuously for 3-5 minutes;

[0015] The detection position is close to the water flow out direction end of the culture area, and the distance from the edge of the water flow out direction end of the culture area is 10cm-20cm.

[0016] The device used in the method for evaluating acute temperature stress of flatfishes comprises a breeding area, a filter, a water storage area and a water pump; the detection site is arranged on the water outflow side of the breeding area; the water inflow end of the breeding area is connected with the valve through a pipeline, and the water outflow end of the breeding area is connected with the filter through a pipeline; the inlet and outlet of the water storage area are connected with the filter and the water pump through pipelines.

[0017] The distance between the water inflow end edge and the water outflow end edge of the breeding area is at least 1 m, and the area of the breeding area needs to ensure that the experimental fishes are not overlapped after being put in.

[0018] The method for evaluating acute temperature stress of flatfishes is used for screening temperature-tolerant individuals of flatfishes.

[0019] Specifically, the first 10% of the total number of flatfishes brought to the detection site by the water flow are temperature-intolerant individuals, and the last 10% of the total number of flatfishes brought to the detection site by the water flow are temperature-tolerant individuals.

[0020] The method for evaluating acute temperature stress of flatfishes is used for screening temperature-tolerant families of flatfishes.

[0021] Specifically, the same number of individuals of flatfishes of different families are marked with different colors of fluorescent tail fins, and then put into the breeding area, and the temperature tolerance or tolerance time of the flatfish individuals is evaluated according to the method; when the total number of half of the flatfishes is brought to the detection site by the water flow, the temperature is recorded as the half-effect temperature, and the time is recorded as T0; the water temperature is kept at the half-effect temperature, the tolerance time T s of the flatfishes brought to the detection site by the water flow before T0 is T i , and the tolerance time T r of the flatfishes brought to the detection site by the water flow after T0 is T i , wherein T i is the tolerance time of the i-th tail fish; one-way ANOVA is performed according to T s of each individual in each family, and the family with T s significantly higher than T s of all individuals in the family, or the family with parents being the wild population and significantly higher than the control group is a non-temperature-tolerant family (P<0.05); one-way ANOVA is performed according to T r of each individual in each family, and the family with T r significantly higher than T s of all individuals in the family, or the family with parents being the wild population and significantly higher than the control group is a temperature-tolerant family (P<0.05).

[0022] Or, the same number of individuals of different families of flatfish are put into the breeding area after being marked with different colors of fluorescent tail fins, and the method is used to evaluate the temperature tolerance of the flatfish individuals; one-way ANOVA is performed on the temperature tolerance of all the individuals of each family, and the temperature tolerance of each family is calculated and compared; in high-temperature stress, the family with a significantly higher temperature tolerance is the high-temperature-tolerant family, and the family with a significantly lower temperature tolerance is the low-temperature-tolerant family (P<0.05); in low-temperature stress, the family with a significantly lower temperature tolerance is the low-temperature-tolerant family, and the family with a significantly higher temperature tolerance is the high-temperature-tolerant family (P<0.05).

[0023] During the experiment, the indexes such as dissolved oxygen, pH, illumination and ammonia nitrogen are kept in the suitable range of the experimental fish, so as to reduce the stress caused by other factors.

[0024] The advantages and positive effects of the present application are as follows:

[0025] 1. The evaluation method of the present application can determine whether the fish can lie flat on the water bottom through water flow, and takes being brought to the detection place as the standard, so as to avoid the stress reaction of the fish caused by manual stirring, and be more objective and accurate.

[0026] 2. The present application firstly calculates the suitable range of water flow speed. The fish in an unbalanced state is simulated by anesthesia, and the minimum flow speed is set to be able to bring all the unbalanced fish to the detection place; the highest flow speed is set to not cause stress to the fish. The flatfish species live by lying flat on the water bottom, and as the water flow speed increases, the fish will change the body posture and muscle tension to resist the impact of the water flow to maintain the state of lying flat on the water bottom. This is a process of consuming energy, which will cause stress to the fish, and the oxygen consumption rate of the fish will also increase. The existing technology mostly uses biochemical indexes such as enzyme activity related to oxidation and immunity in serum and liver to judge whether the stress is large, but this needs to be detected after sample taking, and cannot be continuously and directly observed. The existing research results show that the increase of stress and oxygen consumption rate usually leads to the increase of the respiratory frequency of fish, and based on this, the present application firstly introduces the observation of the respiratory frequency into the evaluation method, so as to more directly judge the stress state of the fish. The respiratory frequency of the main cultured flatfish species such as Paralichthys olivaceus and Scophthalmus maximus is mostly between 30 times / min and 40 times / min when at rest, and therefore, the standard of the highest water flow speed is set to be “30%-50% higher than the average respiratory frequency when at rest”, if it is too low, it is difficult to distinguish whether the respiratory frequency is increased, and if it is too high, it will cause stress to the experimental fish. The suitable flow speed set by the present application can screen out all the unbalanced individuals, and will not cause stress to the fish, so as to ensure that the unbalanced fish are caused by the inability to tolerate the limit temperature, and the result is more accurate.

[0027] 3. In temperature tolerance experiments, especially high-temperature tolerance experiments, if unbalanced or dead experimental fish are not removed promptly, the concentrations of ammonia nitrogen and nitrite nitrogen in the experimental water often become too high. This can cause toxic effects on the remaining fish, often resulting in mass mortality and experimental failure. Furthermore, for fish like flatfish that lie at the bottom, it is difficult to assess their condition. To avoid this, frequent movement and observation are necessary to check for imbalance or death, causing even greater stress. In this invention, unbalanced experimental fish are directly brought to the testing area and can be quickly removed from the water. This reduces the toxic effects on the remaining experimental fish caused by fish mortality and increased concentrations of ammonia nitrogen and nitrite nitrogen, resulting in more reliable results. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of a temperature stress assessment device for flatfish according to an embodiment of the present invention. Detailed Implementation

[0029] The present invention will now be further described in detail with reference to embodiments and accompanying drawings.

[0030] like Figure 1 As shown, the device used in this embodiment of the invention includes: a breeding area, a filter c, a water storage area, and a water pump g; wherein, a detection position is provided on the side of the water flow outward direction of the breeding area; the water flow inward direction end of the breeding area is connected to a valve h through a pipeline, and the water flow outward direction end of the breeding area is connected to the filter c through a pipeline; the inlet and outlet of the water storage area are respectively connected to the filter c and the water pump g through pipelines.

[0031] The breeding area is a long breeding tank a, with dimensions of 2m × 0.8m × 0.4m;

[0032] The detection point is baffle b, and the water storage area is water tank d;

[0033] Baffle b is located near the side of the elongated aquaculture tank a in the direction of water flow out, and is 10cm away from the edge of the elongated aquaculture tank a in the direction of water flow out.

[0034] The water storage tank is equipped with an oxygenation device (e) and a temperature control device (f). A water pump (g) is used to continuously pump water from the water storage tank (d) into the long aquaculture tank (a), and a valve (h) controls the water flow rate. At the location where the experimental fish are placed on the side of the long aquaculture tank (a) in the direction of water flow, sensors (i) for flow rate, temperature, dissolved oxygen, pH, etc., and an underwater camera (j) are installed.

[0035] The function of baffle b is to ensure that only individuals of the flounder that cannot lie flat on the bottom remain at baffle b. If an individual of the flounder that occasionally swims to the detection position in the breeding area touches b, it will swim away. This avoids mistaking individuals of the flounder that occasionally swim to the detection position for individuals that cannot lie flat on the bottom, thus ensuring the accuracy and reliability of the experimental results.

[0036] Example 1 assess the high temperature tolerance temperature or tolerance time of Paralichthys olivaceus

[0037] 1. Set the experimental water flow rate

[0038] (1) Determine the minimum water flow rate

[0039] Take 30 experimental Paralichthys olivaceus (body weight 52±5g), after anesthesia with 100mg / L MS-222, place them on the bottom of the long aquaculture tank (2m x 0.8m x 0.4m), start from 0m / s, gradually increase the flow rate at a rate of 0.1m / s per hour, when the flow rate reaches 0.2m / s, all experimental fish are carried into the detection position baffle of the long aquaculture tank, record 0.2m / s as the minimum water flow rate.

[0040] The water depth during the experiment is 0.3m, the water temperature is 20.2±0.5℃, the DO is ≥6mg / L, the pH is 7.7, the salinity is 32, and the nitrite nitrogen is <0.1mg / L.

[0041] (2) Determine the maximum water flow rate

[0042] Take 30 experimental fish from the same batch, put them into the long aquaculture tank, and acclimate them for 24h at 20.2±0.5℃, then use an underwater camera to observe the respiratory rate of the experimental fish at rest, and calculate the average respiratory rate of the experimental fish at rest to be 32 times / min; then, gradually increase the flow rate from 0.2m / s at a rate of 0.1m / s per hour, and use an underwater camera to observe the respiratory rate of the experimental fish until the flow rate reaches 0.5m / s, at which point the average respiratory rate of the experimental fish is 43 times / min, which is 37.6% higher than the average respiratory rate at rest, record 0.5m / s as the maximum water flow rate.

[0043] (3) Set the experimental flow rate

[0044] The experimental water flow rate is determined to be between the minimum water flow rate and the maximum water flow rate, which is 0.3m / s in this case.

[0045] 2. Assess the temperature tolerance and tolerance time of flatfish

[0046] Take 30 experimental fish from the same batch, put them into the long aquaculture tank, and acclimate them for 24h at 20.2±0.5℃, then set the experimental water flow rate to 0.3m / s, and use a temperature controller to start warming up at a rate of 0.5℃ / h until the temperature reaches 31.0℃, at which point the experimental fish cannot lie flat on the bottom of the water, then gradually increase the temperature at a rate of 0.1℃ / h until the temperature reaches 34.3℃, at which point all experimental fish cannot lie flat on the bottom of the water and are carried into the detection position baffle of the long aquaculture tank;

[0047] When the experimental fish individual is brought into the long-shaped breeding tank detection position baffle by water flow, it is fished out in time, and the temperature or time corresponding to this moment is recorded, that is, the temperature or time of paralichthys olivaceus individual tolerance.

[0048] Example 2 High temperature tolerance temperature screening of paralichthys olivaceus high temperature tolerant individuals

[0049] 50 experimental fish of the same batch as example 1 were put into the experimental device, and the method of example 1 was used. The first 5 experimental fish brought to the detection position baffle by water flow were high temperature intolerant individuals, and their tolerance temperature was 31.8±0.7℃(mean±standard deviation); the last 5 experimental fish brought to the detection position baffle by water flow were high temperature tolerant individuals, and their tolerance temperature was 33.7±0.5℃(mean±standard deviation). By t-test, the tolerance temperature of high temperature tolerant individuals was significantly higher than that of high temperature intolerant individuals(P<0.05), see table 1.

[0050] Table 1 tolerance temperature of 50 paralichthys olivaceus individuals

[0051]

[0052] Example 3 High temperature tolerance time screening of paralichthys olivaceus high temperature tolerant families

[0053] 1, set the experimental water flow rate

[0054] (1) determine the minimum flow rate

[0055] Take 4 paralichthys olivaceus breeding families and the control group of wild paralichthys olivaceus as the parent source, take 5 experimental fish(body weight 72±3g) of each, after being anesthetized with 200mg / L MS-222, place them flat at the bottom of the long-shaped breeding tank(2m×0.8m×0.4m), start from 0m / s and gradually increase the flow rate, when the flow rate is 0.3m / s, all experimental fish are brought into the long-shaped breeding tank detection position baffle by water flow, and record 0.3m / s as the minimum flow rate.

[0056] During the experiment, the water depth was 0.35m, the water temperature was 22.0±0.5℃, the DO was≥6mg / L, the pH was 7.8, the salinity was 31, and the nitrite nitrogen was<0.1mg / L.

[0057] (2) determine the maximum flow rate

[0058] Take the same batch of 4 Paralichthys olivaceus selected family and control group of experimental fish (body weight 72 ± 3 g) each 5, put into the long-shaped aquaculture tank, 22.0 ± 0.5 ℃ under temporary 24 h make it fully adapted to the observation of experimental fish resting respiratory frequency, and calculate the average respiratory rate of experimental fish at rest 35 times / min, then, from 0.3 m / s at a rate of 0.2 m / s per hour gradually improve the flow rate, with underwater camera observation of experimental fish respiratory frequency to 0.7 m / s, the average respiratory rate of experimental fish was 50 times / min, higher than the average respiratory rate at rest increased by 42.9%, record 0.7 m / s as the experimental highest water flow rate.

[0059] (3) set the experimental flow rate

[0060] The experimental water flow rate is determined between the minimum water flow rate and the maximum water flow rate, which is 0.5 m / s this time.

[0061] 2, screening of Paralichthys olivaceus high temperature family

[0062] (1) take the same batch of 4 families and control group of experimental Paralichthys olivaceus (body weight 72 ± 3 g) each 10, with different colors of fluorescent tail fin after put into the experimental device, 22.0 ± 0.5 ℃ temporary 24 h, set the experimental water flow rate of 0.5 m / s, use temperature controller to start warming, at a rate of 0.5 ℃ / h to 31.0 ℃ begin to appear unable to flat water bottom experimental fish, then gradually increase the temperature at a rate of 0.2 ℃ / h, to 32.6 ℃ about half of the experimental fish (24 fish) can't flat water bottom, carried by the water flow to the detection of the back baffle, 32.6 ℃ is the half tolerance temperature, the time is recorded as T0, and the water temperature is kept at this temperature.

[0063] (2) when the experimental fish that can't flat water bottom is carried by the water flow to the back baffle of the tank, it is fished out in time, and the corresponding time is recorded.

[0064] (3) the fish carried by the water flow to the baffle before T0 of the 4 families and control group are 8, 2, 2, 7, 5, respectively, the tolerance time of each fish is calculated according to the formula T s = T0-T i , family 2 and family 3 are too small in quantity, so statistical analysis is not carried out. The tolerance time of family 1, family 4 and control group is 6.8 ± 2.1 h (mean ± standard deviation), 3.7 ± 1.9 h (mean ± standard deviation) and 3.0 ± 2.2 h (mean ± standard deviation) respectively. The t-test analysis result shows that the tolerance time of family 1 is significantly higher than that of the control group (P < 0.05), so family 1 is considered as a high temperature intolerant family.

[0065] (4) the fish fished out after T0 of the 4 families and control group are 2, 8, 8, 3, 5, respectively, the tolerance time of each fish is calculated according to the formula Tr = T i T0 calculation, family 1, 4 quantity is too small, so no statistical analysis. Family 2, family 3 and control group tolerance time were 67.5±5.3h (mean±standard deviation), 49.4±6.8h (mean±standard deviation) and 44.2±7.5h (mean±standard deviation). t-test analysis showed that family 2 tolerance time was significantly higher than that of the control group (P<0.05), so family 2 was considered as a high temperature tolerance family.

[0066] Example 4 Screening of low temperature tolerance temperature of Scophthalmus maximus low temperature tolerance family

[0067] 1. Set the experimental flow rate

[0068] (1) Determine the minimum flow rate

[0069] 5 Scophthalmus maximus families each take 5 experimental fish (body weight 57±4g), after anesthesia with 50mg / L MS-222, place them on the bottom of the long-shaped aquaculture tank (2m×0.8m×0.4m), start from 0m / s and gradually increase the flow rate, when all experimental fish are carried into the long-shaped aquaculture tank detection site baffle by water flow at 0.3m / s, record 0.3m / s as the minimum flow rate.

[0070] During the experiment, the water depth was 0.3m, the water temperature was 18.2±0.4℃, the DO was ≥5.5mg / L, the pH was 7.6, the salinity was 30, and the nitrite nitrogen was <0.1mg / L.

[0071] (2) Determine the maximum flow rate

[0072] Take 5 experimental fish (body weight 57±4g) of the same batch of 5 Scophthalmus maximus families, put them into the long-shaped aquaculture tank, and after 24h of temporary culture at 18.2±0.4℃ to fully adapt, use an underwater camera to observe the respiratory frequency of the experimental fish at rest, and calculate the average respiratory frequency of the experimental fish at rest to be 30 times / min. Subsequently, gradually increase the flow rate at a rate of 0.1m / s per hour from 0.3m / s, and use an underwater camera to observe the respiratory frequency of the experimental fish until 0.6m / s, at which time the average respiratory frequency of the experimental fish is 40 times / min, which is 33.3% higher than the average respiratory frequency at rest. Record 0.6m / s as the maximum flow rate of the experiment.

[0073] (3) Set the experimental flow rate

[0074] The experimental flow rate is determined between the minimum flow rate and the maximum flow rate, which is 0.5m / s in this case.

[0075] 2. Screening of Scophthalmus maximus low temperature tolerance family

[0076] (1) Take 5 families of 10 experimental turbot (body weight 57±4g) from the same batch, mark the tail fins with different colors, and put them into the experimental device. After 24h of temporary culture at 18.2±0.4℃, set the experimental water flow rate to 0.5m / s, and reduce it to 2.0℃ at a rate of 1.0℃ / h to start the experiment. Then gradually reduce the temperature at a rate of 0.1℃ / h, and all experimental fish cannot lie flat on the bottom of the water, and are carried by the water flow to the detection position of the baffle.

[0077] (2) When the experimental fish that cannot lie flat on the bottom of the water are carried to the baffle, they are immediately fished out, and the corresponding temperature is recorded.

[0078] (3) According to the tolerance temperature of all turbot individuals in each family, the statistical analysis result shows that the tolerance temperatures of families 1, 2, 3, 4, and 5 are -1.5±0.5℃ (mean±standard deviation), -1.8±0.3℃ (mean±standard deviation), 0.2±0.4℃ (mean±standard deviation), 1.6±0.5℃ (mean±standard deviation), and -0.3±0.8℃ (mean±standard deviation), respectively. According to single-factor analysis of variance, the tolerance temperatures of families 1 and 2 are significantly lower than the average tolerance temperature of all families -0.4±1.2℃ (mean±standard deviation) (P<0.05), which are recorded as low-temperature-resistant families, and the average tolerance temperature of family 4 is significantly higher than the average tolerance temperature of all families (P<0.05), which is recorded as a low-temperature-resistant family.

[0079] The above is only a preferred embodiment of the present application, and is not intended to limit the present application in other forms. Any skilled person in the art can modify or change the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent change and modification made according to the technical essence of the present application to the above embodiments still fall within the protection scope of the present application.

Claims

1. A method for assessing acute thermal stress in flatfish, characterized by: Placing the flatfish into the breeding area, and after 24 hours of temporary breeding, setting the experimental water flow rate, and increasing or decreasing the temperature at a rate of 0.5-1.0℃ / h until the flatfish can no longer lie flat on the bottom; then continuously increasing or decreasing the temperature at a rate of 0.1-0.2℃ / h until all the flatfish can no longer lie flat on the bottom and are carried into the detection site by the water flow; and setting the detection site on the side of the water flow outlet direction of the breeding area; When the flatfish is carried into the detection site by the water flow, it is immediately fished out, and the temperature or time corresponding to the moment is recorded, i.e., the temperature tolerance or time tolerance of the flatfish.

2. The method for evaluating the temperature acute stress of flatfish according to claim 1, characterized in that: the minimum water flow rate is: placing a plurality of flatfish into the breeding area, gradually increasing the flow rate at a rate of 0.1-0.2 m / s per hour from 0 m / s, and the flow rate at which all the flatfish are carried into the detection site by the water flow is the minimum water flow rate; the maximum water flow rate is: placing a plurality of flatfish into the breeding area, and after 24 hours, recording the average breathing frequency of the flatfish at rest; then gradually increasing the flow rate at a rate of 0.1-0.2 m / s per hour from the minimum flow rate, and recording the real-time average breathing frequency of the flatfish; and the flow rate at which the real-time average breathing frequency is 30-50% higher than the average breathing frequency at rest is the maximum water flow rate.

3. The method for evaluating the temperature acute stress of flatfish according to claim 1, characterized in that: the detection site is close to the end of the water flow outlet direction of the breeding area, and the distance between the detection site and the edge of the end of the water flow outlet direction of the breeding area is 10-20 cm. The method is used for screening temperature-tolerant individuals of flatfish; the first 10% of the total number of flatfish carried into the detection site by the water flow are temperature-intolerant individuals, and the last 10% of the total number of flatfish carried into the detection site by the water flow are temperature-tolerant individuals.

4. Use of a method for assessing acute thermal stress in flatfish according to any one of claims 1 to 3, characterized in that: The method is used for screening temperature-tolerant families of flatfish.

5. Use of a method for assessing acute thermal stress in flatfish according to any one of claims 1 to 3, characterized in that: ​ Specifically, the same number of flatfish from different families were placed in the rearing area after their tail fins were marked with fluorescent markers of different colors. The tolerance temperature or tolerance time of the individual flatfish was assessed using the method described above. The temperature at which half the total number of flatfish were carried to the detection point by the water flow was recorded as the half-maximal effect temperature, and the time was recorded as T0. The water temperature was maintained at the half-maximal effect temperature, and the tolerance time T of the flatfish carried to the detection point before T0 was recorded. s = T0 - T i The tolerance time T of the flounder that was carried to the detection position by the water flow after T0. r = T i -T0, where T i The tolerance time of the i-th fish; based on T for each individual within each family. s Perform a one-way ANOVA, T s The families with significantly higher rates were those that were not heat-tolerant. P <0.05); based on T for each individual within each family lineage r Perform a one-way ANOVA, T r Significantly higher pedigree ( P <0.05) indicates a heat-tolerant family; the same number of individuals from different families were fluorescently labeled with different colors on their tail fins before being placed in the rearing area, and the individual temperature tolerance of the flounder was assessed using the method described above; a one-way ANOVA was performed based on the individual temperature tolerance of all flounder individuals in each family to calculate and compare the temperature tolerance of each family; under high-temperature stress, families with significantly higher and lower temperature tolerance were respectively heat-tolerant and heat-intolerant families (…). P <0.05); In the low-temperature stress experiment, families with significantly lower and higher tolerance to temperature were respectively low-temperature tolerant families and low-temperature intolerant families ( ). P <0.05).

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