Method for evaluating salt tolerance of marine fish based on gill kidney NKA activity change

Abstract

The invention provides a method for evaluating salt tolerance of marine fish based on gill kidney NKA activity change, and belongs to the technical field of aquaculture. A method for evaluating salt tolerance of marine fish based on gill and kidney sodium potassium ATP enzyme activity change comprises the following steps: culturing marine fish fries under to-be-detected salinity, regularly detecting NKA enzyme activity in gill filaments and / or kidney tissues of the marine fish, and when the NKA enzyme activity in the gill filaments and / or kidney tissues in 6-12h of culturing reaches a stable state, judging the salt tolerance of the marine fish. And judging that the marine fish has tolerance to the salinity to be detected, otherwise, judging that the marine fish does not have tolerance. According to the method, the salt tolerance of the marine fish can be accurately evaluated, the evaluation period is short, hysteresis does not exist, meanwhile, the evaluation method needs a small number of samples, only part of tissue samples of the marine fish are taken, sample damage is small, the fry detection cost is greatly reduced, and the method has high market application and popularization value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aquaculture, and particularly relates to a method for evaluating salt tolerance of seawater fish based on changes in gill and kidney Na / K ATP (NKA) enzyme activity. BACKGROUND

[0002] Currently, the detection method for salt tolerance of fish is mainly to calculate the 96h half lethal concentration (LC 50 ) and safe concentration (SC) through acute toxicity experiment or to use long-term indicators such as growth rate and survival rate, which has long cycle, strong hysteresis and large sample loss. In addition, in the actual production process, the cost of seedlings of some economic species (such as Epinephelus and Scophthalmus maximus) is high, and a large number of them used for toxicity experiment will increase the burden of aquaculture enterprises. Compared with the traditional method, the present method has the characteristics of short detection time, high evaluation efficiency and small sample requirement. SUMMARY

[0003] Therefore, the present application aims to provide a novel method for evaluating salt tolerance of seawater fish, which takes seawater fish fry as the object and judges the salt tolerance of seawater fish based on the changes in NKA activity in gills and / or kidneys, and has the characteristics of rapid and accurate detection and low detection cost.

[0004] The present application provides a method for evaluating salt tolerance of seawater fish based on changes in gill and kidney NKA enzyme activity, which comprises the following steps:

[0005] The seawater fish fry is cultured at a to-be-detected salinity, and the NKA enzyme activity in gill filaments and / or kidney tissues of the seawater fish is detected at regular time intervals. When the NKA enzyme activity in gill filaments and / or kidney tissues reaches stability at 6-12h of culture, it is judged that the seawater fish has tolerance to the to-be-detected salinity; otherwise, it does not have tolerance.

[0006] Preferably, the length of the seawater fish fry is 4.5-5.5cm.

[0007] Preferably, the weight of the seawater fish fry is 1.4-2.6g.

[0008] Preferably, the seawater fish includes Epinephelus and / or Pagrus major.

[0009] Preferably, the to-be-detected salinity includes seawater salinity.

[0010] Preferably, the seawater salinity includes 1-20.

[0011] Preferably, the detection at regular time intervals includes detection at 0h, 6h and 12h of culture or detection at 0h, 6h, 12h and 24h of culture.

[0012] Preferably, when the NKA enzyme activity in the gill filament and / or kidney tissue reaches stability at 6h of cultivation, it means that there is no significant difference in the NKA enzyme activity in the gill filament and / or kidney tissue at 6h of cultivation compared with that at 12h of cultivation.

[0013] Preferably, when the NKA enzyme activity in the gill filament and / or kidney tissue reaches stability at 12h of cultivation, it means that there is no significant difference in the NKA enzyme activity in the gill filament and / or kidney tissue at 12h of cultivation compared with that at 24h of cultivation.

[0014] Preferably, the gill filament and / or kidney tissue of the marine fish is sampled in a state of anesthesia of the marine fish.

[0015] The application provides application of the method in aquaculture.

[0016] Preferably, the aquatic product comprises a black seabream and / or a red seabream.

[0017] The cultivation salinity of the black seabream is 10-25.

[0018] The cultivation salinity of the red seabream is 1-25.

[0019] The application provides a method for evaluating the salt tolerance of marine fish based on the change of NKA enzyme activity in gill and kidney, which takes fish fry as the evaluation object, carries out cultivation under a to-be-tested salinity, detects the sodium-potassium ATPase activity in the gill filament and / or kidney tissue of the marine fish at a fixed time, judges that the marine fish has tolerance to the to-be-tested salinity when the NKA enzyme activity in the gill filament and / or kidney tissue reaches stability at 6-12h of cultivation, and judges that the marine fish does not have tolerance otherwise. The application utilizes the preliminary development of the gill and kidney, which are the osmoregulatory organs of the fish fry, and the ion transport capacity and more significant response to salinity fluctuation of the gill and kidney, so as to reflect the influence of salinity stress on fish by the NKA enzyme activity in the gill filament and / or kidney tissue. Experiments show that the salt tolerance concentration obtained by the method for evaluating the conventional marine fish is consistent with the salt tolerance concentration obtained by the 96h acute toxicity experiment, which indicates that the method can accurately evaluate the salt tolerance of marine fish, has a shorter evaluation period, no lag, needs a small amount of samples, only takes part of the tissue samples of the marine fish, and has small sample damage, greatly reduces the detection cost of the fish fry, and has high market promotion and application value. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Fig. 2 shows the NKA activity change results of the black seabream under different salinities, wherein A is the gill tissue measurement result, and B is the kidney tissue measurement result.

[0021] Figure 2 Fig. 3 shows the NKA activity change results of the red seabream under different salinities, wherein A is the gill tissue measurement result, and B is the kidney tissue measurement result. DETAILED DESCRIPTION

[0022] The present invention provides a method for evaluating the salt tolerance of marine fish based on the change of gill nephrase activity, comprising the following steps: culturing marine fish fry under a salinity to be tested, regularly detecting the gill nephrase activity in the gill filaments and / or kidney tissues of the marine fish, and judging that the marine fish has tolerance to the salinity to be tested when the gill nephrase activity in the gill filaments and / or kidney tissues reaches stability after culturing for 6 to 12 hours; otherwise, the marine fish has no tolerance.

[0023] In the present invention, the length of the marine fish fry is preferably 4.5 to 5.5 cm, and can be 5 cm. The weight of the marine fish fry is preferably 1.4 to 2.6 g, and can be 1.6 to 2.4 g, or 1.8 to 2.2 g, and can also be 2.0 g. The marine fish preferably include sea bass and / or black sea bream. The osmotic pressure regulating organs (such as gills, kidneys, intestines, etc.) of the marine fish fry have been initially developed, and they have basic ion transport capabilities, but the regulation efficiency is still lower than that of adult fish. The developmental stage of the marine fish fry responds more significantly to salinity fluctuations and can more intuitively reflect the impact of salinity stress on fish. In aquaculture, seedling cultivation is a key link in salinity adaptability research. The marine fish fry of the above specifications are usually in the acclimatization stage before stocking or the initial stage of pond farming, and their salinity tolerance directly affects the seedling survival rate and farming efficiency. Therefore, using the above-mentioned specifications of marine fish fry as an evaluation method can not only screen for suitable salinity, but also evaluate the survival rate of fry under the tolerated salinity in advance, thereby improving breeding efficiency.

[0024] In the present invention, the salinity to be measured preferably includes a salinity lower than seawater salinity. The salinity lower than seawater salinity preferably includes 1 to 20, can be 3 to 15, or can be 5 to 10. Since my country is rich in saline-alkali land resources, saline-alkali water can be used for aquaculture. However, the salinity in saline-alkali water aquaculture is a key factor affecting the survival rate, growth performance, metabolic level, nonspecific immune function and osmotic pressure regulation ability of fish. Therefore, screening the salt-tolerant concentration suitable for the survival of marine fish from salinities lower than seawater can lay the foundation for aquaculture in saline-alkali water.

[0025] The present invention does not particularly limit the parameters for the aquaculture process, and conventional marine fish aquaculture conditions known in the art may be employed. For example, the dissolved oxygen content of the water is 7.50-8.1 mg / L, the pH is 8.10-8.7, the ammonia nitrogen content is 0.09-0.11 mg / L, and the water temperature during the experiment is 20.5-25°C.

[0026] In the present invention, the gill nephrase activity in the gill filaments and / or kidney tissues of marine fish is used as a screening index for salt tolerance. The survival of marine fish at different salinities depends on their own osmotic regulation ability, and the gills and kidneys are the main tissues for osmotic regulation. The NKA enzyme is the main driving force for ion transport, and its activity change is related to the osmotic regulation ability. When marine fish are transferred from a hypertonic environment to a hypotonic environment, the NKA enzyme activity in their gill tissues shows a typical adaptive regulation pattern. Under hypotonic stress, the enzyme activity is significantly increased, enhancing the ion absorption capacity of the fish and helping to maintain the plasma osmotic pressure within an appropriate physiological range. In addition, as the main driving force for ion transport, the activity change of the NKA enzyme directly determines the osmotic regulation ability of the kidney. Therefore, by monitoring the time when the fish plasma ion concentration and tissue NKA activity reach stability, its salinity tolerance performance can be quickly assessed.

[0027] In the present invention, described timing detection is preferably included in the detection when cultivating the 0th, 6th and 12th hour or detects when cultivating the 0th, 6th, 12th and 24th hour.When the gill nephrase activity in the gill filaments and / or kidney tissue measured when cultivating the 6th hour reaches stability, preferably refers to that compared with cultivating the 12th hour, the gill nephrase activity in the gill filaments and / or kidney tissue had no significant difference when cultivating the 6th hour.When the gill nephrase activity in the gill filaments and / or kidney tissue measured when cultivating the 12th hour reaches stability, preferably refers to that compared with cultivating the 24th hour, the gill nephrase activity in the gill filaments and / or kidney tissue had no significant difference when cultivating the 12th hour.When the gill nephrase activity in the gill filaments and / or kidney tissue measured when cultivating the 6th hour reaches stability, preferably refers to that compared with cultivating the 24th hour, the gill nephrase activity in the gill filaments and / or kidney tissue had no significant difference when cultivating the 12th hour. When the NKA enzyme activity in the gill filaments and / or kidney tissues does not reach stability after 6 hours of cultivation, the NKA enzyme activity in the gill filaments and / or kidney tissues after 24 hours of cultivation is continued to be measured. When the NKA enzyme activity in the gill filaments and / or kidney tissues does not reach stability after 12 hours of cultivation, it is judged that the marine fish fry cannot adapt to growth under the salinity to be measured. It is preferred to use the NKA enzyme activity in the gill filaments and kidney tissues as evaluation indicators at the same time to help obtain more accurate and reliable evaluation results. When the salt tolerance concentrations obtained by the two evaluation indicators are inconsistent, when the salinity of the sample to be tested is lower than that of seawater, the highest salt tolerance concentration screened is used as the tolerant salinity.

[0028] In the present invention, the gill filaments and / or kidney tissue of the marine fish are preferably sampled while the marine fish is anesthetized. Anesthesia sampling is beneficial to reducing damage to the marine fish and keeping the test subject alive.

[0029] The present invention provides application of the method described in the above technical solution in aquaculture.

[0030] In the present application, the aquatic products include black seabream and / or parrotfish; the breeding salinity of the black seabream is preferably 10-25, and can be 12, 15, 18, 20 and 22; the breeding salinity of the parrotfish is 1-25, and can be 3, 5, 7, 10, 12, 15, 18, 20 and 22.

[0031] In one embodiment of the present application, the method developed in the present application is used to test the salt tolerance of black seabream, and the results show that the black seabream fry has good tolerance to salinity above 10, which is consistent with the acute toxicity test results. In another embodiment of the present application, the method developed in the present application is used to test the salt tolerance of parrotfish, and the results show that the parrotfish fry has good tolerance to salinity above 1, which is also consistent with the acute toxicity test results.

[0032] The method for evaluating the salt tolerance of marine fish based on the NKA activity of tissues provided by the present application is described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0033] Example 1

[0034] A method for evaluating the salt tolerance of black seabream based on the NKA activity of tissues

[0035] Black seabream fry with a size of 5 cm (body length: 5.00±0.50 cm; body weight: 2.50±0.54 g) were selected for the experiment. Before the experiment, the two types of fish were transferred to 1500 L temporary holding tanks, and the water for breeding was prepared by adding seawater crystals to aerated tap water to ensure that the water quality met the experimental requirements. During the temporary holding period, the environmental water temperature was controlled at 25.50±0.50℃, the dissolved oxygen was maintained at 8.50 mg / L, the pH value was 7.93±0.12, and the ammonia nitrogen concentration was less than 0.15 mg / L. To keep the water clean, 80% of the water was replaced every day, and feed was fed twice at regular intervals to ensure the health of the fry and the accuracy of the experiment.

[0036] Five salinity gradients were set in the experiment, including natural seawater (control group, salinity 25) and four experimental groups with salinities of 10, 5, 3 and 1. Three parallel groups were set up under each salinity gradient, with 30 fish in each group. The experimental containers were circular plastic tanks with a diameter of 100 cm and a volume of 200 L. The experimental water was prepared by mixing seawater crystals and filtered tap water in proportion and calibrated by a salinity meter to ensure the accuracy of the salinity. During the experiment, feed was fed twice at regular intervals every day, and oxygen was continuously supplied with a dissolved oxygen content of (7.80±0.30) mg / L. In addition, the pH value of the water was 8.40±0.30, the ammonia nitrogen concentration was controlled at (0.1±0.01) mg / L, and the water temperature was maintained between 20.5-25℃ to ensure the stability of the experimental conditions.

[0037] During the salinity experiment, nine fish were randomly selected from each group as samples at the start (0 h) and at 6, 12, 24, 48, 72, and 96 h of the experiment. The fish were anesthetized with 10 mg / L MS-222 (anesthesia time was controlled within 30 s) and then placed on ice. A certain amount of gill and kidney tissue samples were collected, added to 9 volumes (w / v) of pre-chilled 0.86% saline, and thoroughly homogenized using a homogenizer at 0°C. The homogenized samples were centrifuged at 4000 rpm for 10 min in a refrigerated centrifuge, and the supernatant was used to measure NKA enzyme activity. Enzyme activity was determined using the Nanjing Jiancheng NKA enzyme assay kit, while protein content was analyzed using the Nanjing Jiancheng total protein assay kit. NKA enzyme activity is defined as the amount of inorganic phosphorus produced by the breakdown of ATP per mg of protein per hour (1 μmol / (mg·h)).

[0038] The experimental results are shown in Figure 1 .Depend on Figure 1 The results show that after black sea bream fry were placed in different salinity groups, their NKA enzyme activity reached stability within 12 hours in the salinity 10 group, but took 24 hours to stabilize in the salinity 1, 3, and 5 groups. Kidney NKA activity also reached stability within 12 hours in the salinity 10 group, 24 hours in salinity 3 and 5, and 72 hours in salinity 1. This indicates that black sea bream have good adaptability and tolerance to salinity 10, suggesting that black sea bream can be cultured in low-salinity conditions at salinity 10.

[0039] Comparative Example 1

[0040] 96h acute toxicity test on black sea bream

[0041] The 96-hour survival rate of black sea bream fry (body length: 5.00±0.50cm; weight: 2.50±0.54g) was measured in a gradient salinity treatment of 10, 5, 3, and 1. Three replicates were set for each gradient, with 20 fish per replicate. The water was aerated continuously throughout the experiment to maintain a dissolved oxygen level of 7.80±0.30mg / L, a pH of 8.40±0.30, and an ammonia nitrogen level of (0.1±0.01)mg / L. The water temperature was maintained at 20.5-25°C. Fish mortality was determined as follows: complete cessation of gill cover or mouth movement, and no contraction of the gill filaments when gently touched with a glass rod.

[0042] The results show that the survival rate of the black seabream fry is 100% under the salinity of 25, 10 and 5. Although no black seabream fry dies after 96 hours of stress in the water body with the salinity of 5, a small number of black seabreams appear to be slow in swimming, lie on the bottom and be more inclined to move in the lower layer of the tank, which indicates that the body responds to the imbalance of the environmental osmotic pressure and is an intuitive signal of physiological function disorder, and indicates that the environment with the salinity of 5 has potential damage to the body. When the black seabream fry is transplanted into the water body with the salinity of 5, acclimation culture is needed to restore the vitality.

[0043] Therefore, it is known that the 96h acute toxicity experiment needs a long time and a large amount of sample loss, and cannot timely indicate the tolerance of the target fish to the salinity of the water body and accurately show the physiological regulation process of the fish body. The 96h acute toxicity experiment result shows that the survival rate of the black seabream fry reaches 100% under the salinity of 25 and 10, and the vitality meets the requirements of low-salt culture, which is completely consistent with the conclusion of Example 1. By comparison, it is known that the application can quickly and accurately detect the tolerance of the seawater fish to low salt by detecting the NKA activity stabilization time of the gill kidney tissue, and the sample loss is small, thereby reducing the economic loss.

[0044] Example 2

[0045] A method for evaluating the salt tolerance of Lateolabrax japonicus based on tissue NKA activity

[0046] Lateolabrax japonicus is selected as the experimental object, wherein the size of the Lateolabrax japonicus is 5cm (body length: 5.00±0.50cm; body weight: 2.00±0.60g), and is cultured under the conditions of the salinity of 10, 5, 3 and 1. The method for evaluating the salt tolerance of Lateolabrax japonicus based on tissue NKA activity is carried out according to the method of Example 1.

[0047] The results are shown in Figure 2 . It is known from Figure 2 that under the environmental stress of the salinity of 10, 5, 3 and 1, the NKA enzyme activity of the gill and kidney tissue of the Lateolabrax japonicus fry tends to be stable within 6-12 hours, which indicates that the Lateolabrax japonicus has good tolerance to the four salinities.

[0048] Comparative Example 2

[0049] 96h acute toxicity experiment of Lateolabrax japonicus

[0050] Using Lateolabrax japonicus fry (body length: 5.00±0.50cm; weight: 2.50±0.54g), seven salinity gradients were set: 0.20 (freshwater), 1, 3, 5, 10, 15, and 25. Each gradient had three replicates, with 20 fish per replicate. During the experiment, water was aerated continuously to maintain dissolved oxygen at 7.80±0.30mg / L, pH at 8.40±0.30, and ammonia nitrogen at (0.1±0.01)mg / L. The water temperature was maintained at 20.5-25°C. After the experiment, the median lethal salinity and safe salinity were calculated using the probit method.

[0051] The results showed that the survival rate of L. japonicus in freshwater was 95%, and the survival rate was 100% after 96 hours in other salinities. This is consistent with the conclusion of Example 2, indicating that L. japonicus is a low-salinity tolerant marine fish species and can be used for aquaculture under low-salinity conditions.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for evaluating salt tolerance of marine fish based on changes in gill-kidney sodium-potassium ATPase activity, characterized in that: The following steps are involved: Marine fish fry are cultured at a salinity to be tested, and the sodium-potassium ATPase activity in the gill filaments and / or kidney tissues of the marine fish is regularly detected. When the sodium-potassium ATPase activity in the gill filaments and / or kidney tissues reaches stability during the 6th to 12th hour of culture, it is determined that the marine fish has tolerance to the salinity to be tested; otherwise, it does not have tolerance.

2. The method according to claim 1, characterized in that The length of the sea fish fry is 4.5 to 5.5 cm; The body weight of the marine fish fry is 1.4 to 2.6 g.

3. The method according to claim 1, characterized in that The marine fish includes sea bass and / or black sea bream.

4. The method according to claim 1, characterized in that The salinity to be measured includes salinity lower than seawater salinity.

5. The method according to claim 4, characterized in that: The salinity lower than seawater includes salinity 1-20.

6. The method according to claim 1, characterized in that The timed detection includes detection at 0h, 6h and 12h of breeding or detection at 0h, 6h, 12h and 24h of breeding.

7. The method according to claim 7, characterized in that: When the sodium-potassium ATPase activity in the gill filaments and / or kidney tissue reaches stability after 6 hours of culture, it means that there is no significant difference in the sodium-potassium ATPase activity in the gill filaments and / or kidney tissue at 6 hours of culture compared with 12 hours of culture; When the sodium-potassium ATPase activity in the gill filaments and / or kidney tissue reaches stability after 12 hours of culture, it means that there is no significant difference in the sodium-potassium ATPase activity in the gill filaments and / or kidney tissue after 24 hours of culture.

8. The method according to any one of claims 1 to 7, characterized in that: The gill filaments and / or kidney tissues of the marine fish are sampled while the marine fish is in an anesthetized state.

9. Use of the method according to any one of claims 1 to 8 in aquaculture.

10. The use according to claim 9, characterized in that: The aquatic products include black sea bream and / or spotted sea bass; The black sea bream is cultured at a salinity of 10 to 25; The culture salinity of the sea bass is 1 to 25.

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