Research method for high-temperature and low-salt tolerance of coelomactra antiquata in different development stages

Abstract

The invention discloses a method for researching high temperature and low salt tolerance of coelomactra antiquata in different development stages, which comprises the following steps: S1, selecting materials of coelomactra antiquata in different development stages, including larvae, juvenile mollusks and young mollusks; s2, performing survival research on materials of coelomactra antiquata in different development stages under acute and chronic temperature rise conditions, and recording the survival rate; s3, studying the survival of the coelomactra antiquata materials in different development stages under the chronic low-salt condition, and calculating the survival rate of the coelomactra antiquata materials. The invention researches the high temperature and low salt tolerance of coelomactra antiquata in different development stages. Results show that two parameters of 192LT50 in a chronic temperature rise process and 96LT50 in an acute temperature rise process have significant differences in different development stages of coelomactra antiquata; the difference of the 192LT50 in different development stages of coelomactra antiquata in the chronic low-salt process is not obvious. The discovery is applied to high-temperature and low-salt domestication of coelomactra antiquata, promotes metamorphosis of larvae, improves the ingestion ability of juvenile mollusks and young mollusks, and assists artificial breeding, breeding, enhancement and release and resource protection of coelomactra antiquata.

Description

Technical Field

[0001] The invention relates to the technical field of artificial cultivation of thuja sutchuenensis, in particular to a method for studying the high temperature and low salt tolerance of thuja sutchuenensis at different developmental stages. Background Art

[0002] The Chinese mullet is a marine commercial shellfish that can be used as both food and medicine. In recent years, artificial breeding of the Chinese mullet has been successful along the coast of Zhanjiang, and large-scale reproduction and release in the nearshore sea area has been carried out. The summer along the coast of Zhanjiang is hot and rainy. The continuous high temperature and heavy precipitation cause the nearshore seawater temperature to continue to rise and the salinity to drop significantly, posing a great threat to the adult Chinese mullet.

[0003] The intertidal zone is located at the junction of water and land. Its environment is affected by both the ocean and the land. It is one of the harshest habitats on the earth. This also causes the intertidal zone ecosystem to be subjected to multi-factor stress. For example, in summer, the intertidal zone is exposed to the sun for a long time during low tide, absorbing a large amount of solar radiation heat, causing its temperature to rise sharply, while a large amount of rainfall will dilute the salinity of the intertidal zone, causing the salinity to drop sharply. Therefore, many scholars have conducted research on the effects of high temperature and low salinity on the survival of bivalve shellfish. Compared with the common population, the survival rate of the disease-resistant strains of one-year-old and two-year-old long oysters (Crassostrea gigas) increased by 5% after 10 days of high temperature stress, and there was no significant difference in the survival rate of the disease-resistant strains of one-year-old long oysters after 10 days of low salt stress, while the survival rate of the two-year-old shellfish decreased by 5%. In the tolerance test of Haliotis diversicolor to high water temperature and low salinity, the acute high temperature that the diversicolor abalone can tolerate is 32℃, the chronic high temperature is 36℃, the acute low salt is 16, and the chronic low salt is 14. In the immune response test of the interaction between temperature (21, 30℃) and salinity (7, 15, 32) of the Philippine clam (Ruditapes philippinarum), it was found that when the temperature increased and the salinity decreased, the immune ability of the shellfish would be seriously affected, resulting in the death of a large number of shellfish. In recent years, the research team has successfully carried out artificial breeding of the Chinese tongue in the coastal area of ​​Zhanjiang, but the tolerance of the Chinese tongue to high temperature and low salt in some areas is still unclear. Since the 1960s, many scholars in Liaoning, Shandong, Fujian and other places have successively carried out artificial breeding research on the Chinese tongue, but the core technology of large-scale Chinese tongue breeding is still difficult to break through. Summary of the invention

[0004] In order to solve the deficiencies in the prior art, the present invention studies the effects of high temperature and low salt on the development of the tongue of the Chinese tuna at different stages, obtains the tolerance of the tongue of the Chinese tuna to high temperature and low salt, and conducts research on the survival of the tongue of the Chinese tuna at different growth stages under different high temperature and low salt stresses, and obtains the semi-lethal temperature (96hLT) of the tongue of the Chinese tuna larvae, juveniles and young shellfish under 96h acute temperature rise. 50) and the half-lethal temperature of 192h chronic heating (192hLT 50 ) and the half-lethal salinity of chronic low salt (192hLS 50 ). This study aims to explore the high temperature and low salt tolerance of Chinese trevally at different developmental stages, and to promote the artificial breeding, cultivation, proliferation and release, and resource protection of Chinese trevally.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for studying the high temperature and low salt tolerance of Chinese taro at different developmental stages, which comprises the following steps:

[0007] S1. Material selection at different developmental stages of the Chinese tongue, including larvae, juveniles and young shellfish;

[0008] S2. Study the survival of the materials of the Chinese tongue at different developmental stages under acute and chronic warming conditions and record the survival rate;

[0009] S3. Study the survival of materials of Chinese tongue at different developmental stages under chronic low-salt conditions and calculate the survival rate.

[0010] In some possible implementations, in step S1:

[0011] Larvae: Shade-drying and running water were used to stimulate 3-4-year-old broodstock to release sperm and eggs. After the fertilized eggs were formed, they were hatched in the original pond. Then, the larvae were cultured in a closed breeding mode until the shell top was swollen and slightly tilted to the front, the front edge of the shell was truncated, the rear edge was round, and the feet were rod-shaped and could be extended out of the shell. This marked that the larvae entered the late stage of the shell top disc larvae. The larvae at this time were used as materials, and their specifications were shell length 246±8.20μm, shell height 230±7.75μm;

[0012] Juvenile spat: When the spat has an outlet and inlet tubes and the foot is axe-shaped, it indicates that the spat has entered the double water tube stage. The spat at this time is used as the material, and its specifications are shell length: 750±50.86μm, shell height: 660±45.32μm;

[0013] Juvenile oysters: After about 45 days of intermediate cultivation, the juvenile oysters (2mm) can be taken out of the pond and released into the sea. The juvenile oysters at this time are used as materials, with the specifications of shell length: 2.15±0.07cm, shell height: 2.04±0.03cm.

[0014] In some possible implementations, step S2 is specifically as follows:

[0015] S2.1. Under acute temperature rise conditions, five temperature gradients were set at 26°C (control group), 28°C, 30°C, 32°C, and 34°C, and three parallel experiments were set for each gradient. The experiment was carried out in a beaker or a plastic bucket. Then seawater was added. The beaker or plastic bucket was temperature-controlled in a water bath or directly heated using an automatic temperature control heating rod. The larvae density was 3 / mL, the juvenile density was 1 / mL, and the juvenile density was 40 / L. The salinity was maintained at 27-28 and the pH was 7.9-8.1. The three developmental stages of the larvae, juveniles, and juveniles of the larvae of the larvae were respectively carried out as described above.

[0016] S2.2. Under chronic warming conditions, start from the temporary culture temperature (26°C), increase at a gradual rate of 1°C / d to 34°C in a beaker or a plastic bucket. Add seawater to the beaker or plastic bucket, and use an automatic temperature control heating rod to control the temperature of the beaker or plastic bucket in a water bath or directly heat it. Then put in the Chinese tongue with a larval density of 3 / mL, a juvenile density of 1 / mL, and a juvenile density of 40 / L. Maintain the salinity at 27-28 and the pH at 7.9-8.1. Experiments were carried out in the three developmental stages of Chinese tongue larvae, juveniles, and juveniles as described above.

[0017] In some possible implementations, step S3 is specifically as follows:

[0018] S3.1. Starting from the temporary culture salinity (28‰), the salinity was gradually reduced at a rate of 2‰ / d until it reached 12‰. The experiment was set up in three parallels and was carried out in one of the beakers or plastic buckets. Seawater was added to the beakers or plastic buckets to maintain the water temperature at 25-26℃ and the pH at 7.9-8.1. The larvae density was 3 / mL, the spat density was 1 / mL, and the juvenile density was 40 / L. The experiments were carried out in the three developmental stages of the larvae, juveniles, and juveniles of the Chinese tongue respectively according to the above.

[0019] S3.2, then calculate the survival rates of the three developmental stages of the larvae, juveniles and young oysters under chronic low-salinity conditions;

[0020] S3.3. The data were expressed as "mean ± standard deviation (X ± SD)". The measurement indicators were processed using Excel 2019 software. SPSS22.0 software was used to perform one-way analysis of variance on the data. The Duncan's multiple comparison method was used to test the significance of differences between different treatments.

[0021] In some possible implementations, in step S3.1, when the three developmental stages of the larvae, juveniles and young shellfish are studied under chronic low-salinity conditions, the algae density in the water body is maintained at (1-2)×10 4 cell / mL、(2-3)×10 4 cell / mL and (5-10)×10 4cell / mL.

[0022] In some possible implementations, the calculation formula of step S3.2 is as follows:

[0023] Actual survival rate formula: SR = N t / N0×100%

[0024] Where, N0 is the initial density / number of Xishi tongue, and Nt is the final density / number of Xishi tongue;

[0025] Relative survival rate formula: P = SR / SR' control group × 100%

[0026] In the formula, SR is the actual survival rate, and SR' control group is the survival rate of the corresponding control group;

[0027] The half-lethal temperature formula is: LT 50 =T1+[(P1-50%) / (P1-P2)]×(T2-T1)

[0028] Where: T1 and T2 are the high and low end temperatures where the survival rate is close to 50%; P1 and P2 are the corresponding relative survival rates;

[0029] The half-lethal salinity formula is: LS 50 =S1+[(P1-50%) / (P1-P2)]×(S2-S1)

[0030] Where: S1 and S2 are the high and low end salinities with a survival rate close to 50%; P1 and P2 are the corresponding relative survival rates.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention studies the high temperature and low salt tolerance of Chinese tongue at different developmental stages. The results show that 192LT 50 and 96LT during acute warming 50 The two parameters of the 192LT in different developmental stages of the shishi tongue were significantly different; 50 There is no significant difference in the different developmental stages of the Chinese tongue. This discovery can be applied to the high temperature and low salt acclimation of the Chinese tongue, promote the metamorphosis of larvae, improve the feeding ability of juveniles and young shellfish, and help the resource protection and proliferation and release of the Chinese tongue. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the survival rate curve of Glossina truncatula larvae under acute high temperature stress for 96 hours;

[0034] Figure 2 This is the survival rate curve of juvenile spat of Ophiopogon japonicus under acute high temperature stress for 96 hours;

[0035] Figure 3 This is the survival rate curve of the juvenile oysters of the Chinese giant oyster shellfish under acute high temperature stress for 96 hours;

[0036] Figure 4 This is the survival rate curve of Glossina truncatula larvae under chronic high temperature stress for 192 hours;

[0037] Figure 5 This is the survival rate curve of juvenile Sparus truncatula under chronic high temperature stress for 192 hours;

[0038] Figure 6 This is the survival rate curve of juvenile oysters of the Chinese oyster shellfish under chronic high temperature stress for 192 hours;

[0039] Figure 7 This is the survival rate curve of Glossina truncatula larvae under chronic low-salinity stress for 192 hours;

[0040] Figure 8 This is the survival rate curve of juvenile Japanese spat under chronic low-salinity stress for 192 hours;

[0041] Fig. 9 This is the survival rate curve of young clams under chronic low-salinity stress for 192 hours. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] The present invention provides a method for studying the high temperature and low salt tolerance of Chinese taro at different developmental stages, which comprises the following steps:

[0044] S1. Material selection at different developmental stages of the Chinese tongue, including larvae, juveniles and young shellfish;

[0045] S2. Study the survival of the materials of the Chinese tongue at different developmental stages under acute and chronic warming conditions and record the survival rate;

[0046] S3. Study on the survival of materials of different developmental stages of Xishi Tong under chronic low-salinity conditions and calculate the survival rate;

[0047] The details are as follows:

[0048] Effects of acute and chronic hyperthermia on survival

[0049] larva

[0050] Preliminary experiments showed that all larvae of Glossina truncatula died within 17 hours at a high temperature of 35°C. Therefore, the acute temperature increase experiment set five temperature gradients of 26°C (control group), 28°C, 30°C, 32°C, and 34°C, with three parallels for each gradient. The experiment was conducted in a 1L beaker, 800mL of seawater was added, and an automatic temperature control heating rod was used for water bath temperature control to keep the initial density of larvae in the water at 3 / mL. During the experiment, Chlorella was fed once in the morning and evening every day to keep the algae density in the water at (1-2)×10 4 cell / mL, maintain salinity at 27-28, pH at 7.9-8.1, change water by 50% isothermally every day, and aerate continuously in microwave form. Take samples every 24 hours (4 times in total), stir the larvae evenly before each sampling, take 20mL of samples with a pipette, count the density of the larvae, repeat three times, and calculate the average value, which is used as the density of the Chinese tongue at the stress time point.

[0051] The experiment started with a temporary culture temperature (26°C) and increased at a gradual rate of 1°C / d until it reached 34°C. Three parallel experiments were set up and the experiment was conducted in a 1L beaker. 800mL of seawater was added to keep the initial density of larvae in the water at 3 / mL. An automatic temperature control heating rod was used to control the water bath temperature. During the experiment, Chlorella was fed once in the morning and evening every day to keep the algae density in the water at (1-2)×10 4 cell / mL, maintain salinity at 27-28, pH at 7.9-8.1, change water by 50% isothermally every day, and continuously aerate in microwave form. Take samples every 48 hours (4 times in total), stir the larvae evenly before each sampling, take 20mL of samples with a pipette, count the density of the larvae, repeat three times, and calculate the average value, which is used as the density of the Chinese tongue at the stress time point.

[0052] Juvenile

[0053] Preliminary experiments showed that all juvenile spat of the Chinese oyster shellfish died within 17 hours at a high temperature of 35°C. Therefore, the acute temperature increase experiment set up five temperature gradients of 26°C (control group), 28°C, 30°C, 32°C, and 34°C, with three parallels for each gradient. The experiment was conducted in a 1L beaker, 800mL of seawater was added, and an automatic temperature control heating rod was used for water bath temperature control to keep the initial density of juvenile spat in the water at 1 / mL. During the experiment, Chlorella was fed once in the morning and evening every day to keep the algae density in the water at (2-3)×10 4 cell / mL, maintain salinity at 27-28, pH 7.9-8.1, change water by 50% isothermally every day, and continuously aerate in microwave mode. Take samples every 24 hours (4 times in total). Before each sampling, stir the spat evenly, take 20mL of samples with a pipette, and count the density of the spat. Repeat three times and calculate the average value, which is used as the density of the tongue at the stress time point.

[0054] The experiment started with a temporary culture temperature (26°C) and increased at a gradual rate of 1°C / d until it reached 34°C. Three parallel experiments were set up and the experiment was conducted in a 1L beaker or a plastic bucket. 800mL of seawater was added to keep the initial density of juveniles in the water at 1 / mL. An automatic temperature control heating rod was used for water bath temperature control. During the experiment, Chlorella was fed once in the morning and evening every day to keep the algae density in the water at (2-3)×10 4 cell / mL, maintain salinity at 27-28, pH 7.9-8.1, change water by 50% isothermally every day, and aerate continuously in microwave form. Take samples every 48 hours (4 times in total). Before each sampling, stir the spat evenly, take 20mL of samples with a pipette, and count the density of the spat. Repeat three times and calculate the average value, which is used as the density of the tongue at the stress time point.

[0055] Baby Shells

[0056] Preliminary experiments showed that all juvenile clams died within 17 hours at a high temperature of 35°C. Therefore, the acute temperature increase experiment set five temperature gradients: 26°C (control group), 28°C, 30°C, 32°C, and 34°C. The experiment was conducted in a 5L plastic bucket, using an automatic temperature control heating rod for direct heating. Three parallels were set for each gradient, and 200 clams were added to each parallel. During the experiment, Chlorella was fed once in the morning and evening every day to keep the algae density in the water at (5-10)×10 4 cell / mL, maintain salinity at 27-28, pH 7.9-8.1, change water by 50% isothermally every day, and aerate continuously in microwave mode. Check the survival of juvenile clams every 3 hours. If the clams are found to be open and the mantle is not closed after acupuncture, remove the dead individuals in time. Record the survival rate of clams at 24h, 48h, 72h, and 96h.

[0057] The experiment started with a temporary culture temperature of 26°C and increased at a gradual rate of 1°C / d until it reached 34°C. Three parallel experiments were set up, with 200 young mussels added to each parallel. The experiment was conducted in one of the 5L plastic buckets, and direct heating was performed using an automatic temperature control heating rod. During the experiment, Chlorella was fed once in the morning and evening every day to keep the algae density in the water at (5-10)×10 4 cell / mL, maintain salinity at 27-28, pH 7.9-8.1, change water by 50% isothermally every day, and aerate continuously in microwave mode. Check the survival of juvenile clams every 3 hours. If the clams are found to be open and the mantle does not close after acupuncture, remove the dead individuals in time. Record the survival of juvenile clams at 48h, 96h, 144h, and 192h.

[0058] Effects of chronic low salt on survival

[0059] larva.

[0060] The experiment started with a temporary salinity of 28‰ and decreased at a gradual rate of 2‰ / d until it reached 12‰. Three parallel experiments were set up and conducted in a 1L beaker. 800mL of seawater was added to keep the initial larval density in the water at 3 / mL. During the experiment, Chlorella was fed once in the morning and evening every day to keep the algae density in the water at (1-2)×10 4 cell / mL, maintain the temperature at 25-26℃, pH at 7.9-8.1, change 50% of the water with equal salinity every day, and continuously aerate with microwave. Take samples every 24h (8 times in total), stir the larvae evenly before each sampling, take 20mL of samples with a pipette, count the density of the larvae, repeat three times, and calculate the average value, which is used as the density of the Chinese tongue at the stress time point.

[0061] Juvenile

[0062] The experiment started with a temporary culture salinity of 28‰ and then decreased at a gradual rate of 2‰ / d until it reached 12‰. Three parallel experiments were set up and conducted in a 1L beaker. 800mL of seawater was added to keep the initial density of juveniles in the water at 1 / mL. During the experiment, Chlorella was fed once in the morning and evening every day to keep the algae density in the water at (2-3)×10 4 cell / mL, maintain the temperature at 25-26, pH 7.9-8.1, change the water by 50% isothermally every day, and continuously aerate in microwave mode. Take samples every 24 hours (8 times in total). Before each sampling, stir the spat evenly, take 20mL of samples with a pipette, and count the density of the spat. Repeat three times and calculate the average value, which is used as the density of the tongue at the stress time point.

[0063] Baby Shells

[0064] The experiment started with a temporary salinity of 28‰ and decreased at a gradual rate of 2‰ / d until it reached 12‰. Three parallels were set for each gradient, and 200 young mussels were added to each parallel. The experiment was conducted in a 5L plastic bucket. During the experiment, Chlorella was fed once in the morning and evening every day to keep the algae density in the water at (5-10)×10 4 cell / mL, maintain the temperature at 25-26, pH 7.9-8.1, change the water by 50% every day with equal salinity, and aerate continuously in microwave mode. Check the survival of the juvenile oysters every 3 hours. If the oysters are found to be open and the mantle is not closed after acupuncture, remove the dead individuals in time. Record the survival rate of the oysters at 24h, 48h, 72h, 96h, 120h, 144h, 168h, and 192h.

[0065] Survival rate calculation

[0066] Actual survival rate formula: SR = N t / N0×100%

[0067] Where, N0 is the initial density / number of Xishi tongue, and Nt is the final density / number of Xishi tongue;

[0068] Relative survival rate formula: P = SR / SR' control group × 100%

[0069] In the formula, SR is the actual survival rate, and SR' control group is the survival rate of the corresponding control group;

[0070] The half-lethal temperature formula is: LT 50 =T1+[(P1-50%) / (P1-P2)]×(T2-T1)

[0071] Where: T1 and T2 are the high and low end temperatures where the survival rate is close to 50%; P1 and P2 are the corresponding relative survival rates;

[0072] The half-lethal salinity formula is: LS 50 =S1+[(P1-50%) / (P1-P2)]×(S2-S1)

[0073] Where: S1 and S2 are the high and low end salinities with a survival rate close to 50%; P1 and P2 are the corresponding relative survival rates.

[0074] Data processing and analysis

[0075] The experimental data were expressed as "mean ± standard deviation (X ± SD)". The measured indicators were processed using Excel 2019 software. SPSS22.0 software was used for one-way analysis of variance on the data, and the significance of differences between different treatments was tested by Duncan's multiple comparison method.

[0076] Results and Analysis

[0077] Effects of acute hyperthermia on the survival of Cynomorium truncatum

[0078] larva

[0079] Depend on Figure 1It can be seen that under acute high temperature stress, as the experimental time prolonged, the survival rate of the larvae of the western tongue in each experimental group changed significantly (P<0.05), and showed a downward trend. Within 96 hours of stress, there were significant differences in the survival rates of the experimental groups (P<0.05), and there were significant differences in the survival rates of the experimental groups and the control group at 24h, 48h, 72h, and 96h (P<0.05). When the experimental group was 28℃ for 96h, the survival rate of the larvae of the western tongue could be maintained at 60.70%, when the experimental group was 30℃ for 48h, the survival rate of the larvae of the western tongue could be maintained at 59.82%, and when the experimental group was 32℃ and 34℃ for 24h, the survival rates of the larvae of the western tongue could be maintained at 62.45% and 43.62%, respectively. Using the two-point method, the semi-lethal high temperature of the larvae of the western tongue at 96h was 28.90℃.

[0080] Juvenile

[0081] Depend on Figure 2 It can be seen that under acute high temperature stress, as the experimental time prolonged, the survival rate of the juveniles of the Chinese tongue in each experimental group changed significantly (P<0.05), and showed a downward trend. Within 96 hours of stress, there were significant differences in the survival rates of the experimental groups (P<0.05), and there were significant differences in the survival rates of the experimental groups and the control group at 24h, 48h, 72h, and 96h (P<0.05). When the experimental group was 28℃ for 96h, the survival rate of the juveniles of the Chinese tongue could be maintained at 67.49%, when the experimental group was 30℃ for 48h, the survival rate of the juveniles of the Chinese tongue could be maintained at 61.82%, and when the experimental group was 32℃ and 34℃ for 24h, the survival rates of the juveniles of the Chinese tongue could be maintained at 62.58% and 26.10%, respectively. Using the two-point method, the semi-lethal high temperature of the larvae of the Chinese tongue at 96h was 29.14℃.

[0082] Baby Shells

[0083] Depend on Figure 3 It can be seen that under acute high temperature stress, as the experimental time prolonged, the survival rate of the juveniles of the tongue of the Chinese tuna in each experimental group changed significantly (P<0.05), and showed a downward trend. Within 96 hours of stress, there were significant differences in the survival rates of the experimental groups (P<0.05), and there were significant differences in the survival rates of the experimental groups and the control group at 24h, 48h, 72h, and 96h (P<0.05). When the experimental group was 28℃ for 96h, the survival rate of the juveniles of the tongue of the Chinese tuna could be maintained at 71.67%, when the experimental group was 30℃ for 48h, the survival rate of the juveniles of the tongue of the Chinese tuna could be maintained at 46.67%, and when the experimental group was 32℃ and 34℃ for 24h, the survival rates of the juveniles of the tongue of the Chinese tuna could be maintained at 66.67% and 55.00%, respectively. Using the two-point method, the semi-lethal high temperature of the larvae of the tongue of the Chinese tuna at 96h was 29.22℃.

[0084] Effects of chronic hyperthermia on the survival of Cynomorium truncatum

[0085] larva

[0086] Depend on Figure 4 It can be seen that under chronic high temperature stress, as the experimental time prolonged, the survival rate of the larvae of the Chinese tongue changed significantly (P<0.05), and showed a downward trend. Within 192 hours of stress, there were significant differences in survival rates when the temperature was 26℃, 28℃, 30℃, 32℃, and 34℃ (P<0.05), and the survival rates were 90.70%, 59.55%, 22.99%, 2.69%, and 2.61%, respectively. Using the two-point method, the semi-lethal high temperature of the larvae of the Chinese tongue at 192h was 29.39℃.

[0087] Juvenile

[0088] Depend on Figure 5 It can be seen that under chronic high temperature stress, as the experimental time prolonged, the survival rate of juvenile spat of C. truncatum changed significantly (P<0.05) and showed a downward trend. Within 192 hours of stress, there were significant differences in survival rate when the temperature was 26℃, 28℃, 30℃, 32℃, and 34℃ (P<0.05), and the survival rates were 91.98%, 67.64%, 30.08%, 10.08%, and 2.82%, respectively. Using the two-point method, the semi-lethal high temperature of juvenile spat of C. truncatum for 192h was 29.42℃.

[0089] Baby Shells

[0090] Depend on Figure 6 It can be seen that under chronic high temperature stress, as the experimental time prolonged, the survival rate of the juvenile oysters of the Chinese tongue changed significantly (P<0.05) and showed a downward trend. Within 192 hours of stress, there were significant differences in survival rates when the temperature was 26℃, 28℃, 30℃, 32℃, and 34℃ (P<0.05), and the survival rates were 92.00%, 70.00%, 46.67%, 16.67%, and 6.67%, respectively. Using the two-point method, the semi-lethal high temperature of the juvenile oysters of the Chinese tongue at 192h was 30.46℃.

[0091] Effects of chronic low salt intake on the survival of Cynomorium truncatum

[0092] larva

[0093] Depend on Figure 7It can be seen that under chronic low-salinity stress, as the experimental time prolonged, the survival rate of the larvae of the Chinese tongue changed significantly (P<0.05) and showed a downward trend. Within 192 hours of stress, there were significant differences in survival rates when the salinity was 20‰, 18‰, 16‰, 14‰, and 12‰ (P<0.05), and the survival rates were 86.14%, 84.16%, 76.45%, 44.43%, and 24.91%, respectively. Using the two-point method, the semi-lethal low salt of the larvae of the Chinese tongue at 192h was 13.93‰.

[0094] Juvenile

[0095] Depend on Figure 8 It can be seen that under chronic low-salinity stress, as the experimental time prolonged, the survival rate of juvenile oysters of C. truncatum changed significantly (P<0.05) and showed a downward trend. Within 192 hours of stress, there were significant differences in survival rates when the salinity was 16‰, 14‰, and 12‰ (P<0.05), and the survival rates were 71.05%, 42.28%, and 26.63%, respectively. Using the two-point method, the semi-lethal low salt for juvenile oysters of C. truncatum of 192h was 13.71‰.

[0096] Baby Shells

[0097] Depend on Fig. 9 It can be seen that under chronic low-salinity stress, as the experimental time prolonged, the survival rate of young juveniles of the Chinese tongue changed significantly (P<0.05) and showed a downward trend. Within 192 hours of stress, there were significant differences in survival rates when the salinity was 16‰, 14‰, and 12‰ (P<0.05), and the survival rates were 83.63%, 45.23%, and 29.36%, respectively. Using the two-point method, the semi-lethal low salt of young juveniles of the Chinese tongue was 13.89‰ at 192h.

[0098] Analysis on high temperature tolerance of Xishi tongue

[0099] During chronic warming, 192LT of Glossina truncatula larvae 50 is 29.39℃, 192LT of spat 50 is 29.14℃, 192LT for young shellfish 50 is 30.46℃; during the acute temperature rise, the 96LT of the larvae of Glossina truncatula 50 is 28.90℃, 96LT of spat 50 is 29.42℃, 96LT for young shellfish 50 The temperature of Xishi tongue in chronic temperature rise and acute temperature rise is 96LT 50The temperature of the oyster was 29.42℃. It can be concluded from this analysis that the oysters of the same developmental period are more tolerant to chronic warming, which is similar to the research results of the long oyster (Crassostrea gigas), the scallop (Patinopecten yessoensis), and the hairy clam (Scapharca subcrenata). When the oysters of the oysters are subjected to acute warming stress, a large amount of ROS is produced in the body, which then induces antioxidant immune response, synthesizes immune-related enzymes, removes reactive oxygen free radicals in the body, and finally resists the influence of adverse environment. However, when the temperature rises too much, the body has no time to remove the produced ROS, and the immune-related enzymes are denatured and inactivated. The metabolic capacity of the juvenile oysters of the oysters is reduced, and the disease is more likely to break through its immune system and threaten its survival. At the same time, it will also aggravate the oxidative stress response of shellfish, produce a large number of reactive oxygen free radicals, damage shellfish tissues and organs, and even cause death. In the process of chronic warming, as the water temperature rises, the larval cilia movement is strengthened, feeding is more active, and growth is faster, which can achieve the purpose of accelerating the seedling process. However, when the temperature rises to a certain level, it will accelerate the growth of pathogenic microorganisms in the water, increase oxygen consumption, and weaken the feeding activity of the Chinese tongue, resulting in a decrease in its growth rate and survival rate.

[0100] Analysis of low salt tolerance of Chinese yew

[0101] 192LS of Glossina truncatula larvae during chronic low salt 50 is 13.93‰, and the 192LS of spat 50 is 13.71‰, and the 192LS of young shellfish 50 The osmotic pressure of the oyster shellfish changes when it is subjected to salinity stress, stimulating the body to produce oxidative stress, inducing the generation of excessive reactive oxygen free radicals (ROS), and causing a series of oxidative damage (such as DNA damage, enzyme inactivation, lipid peroxidation, etc.). In this process, shellfish need to consume a lot of energy to maintain their own osmotic pressure balance, resulting in a reduction in feeding rate and energy used for growth, and ultimately affecting survival due to long-term insufficient feeding.

[0102] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0103] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for studying the high temperature and low salt tolerance of Chinese taro at different developmental stages, characterized in that: The following steps are involved: S1. Material selection at different developmental stages of the Chinese tongue, including larvae, juveniles and young shellfish; S2. Study the survival of the materials of the Chinese tongue at different developmental stages under acute and chronic warming conditions and record the survival rate; S3. Study the survival of materials of Chinese tongue at different developmental stages under chronic low-salt conditions and calculate the survival rate.

2. The method for studying the high temperature and low salt tolerance of Chinese taro at different developmental stages according to claim 1, characterized in that: In step S1: Larvae: Shade-drying and running water were used to stimulate 3-4-year-old broodstock to release sperm and eggs. After the fertilized eggs were formed, they were hatched in the original pond. Then, the larvae were cultured in a closed breeding mode until the shell top was swollen and slightly tilted to the front, the front edge of the shell was truncated, the rear edge was round, and the feet were rod-shaped and could be extended out of the shell. This marked that the larvae entered the late stage of the shell top disc larvae. The larvae at this time were used as materials, and their specifications were shell length 246±8.20μm, shell height 230±7.75μm; Juvenile spat: When the spat has an outlet and inlet tubes and the foot is axe-shaped, it indicates that the spat has entered the double water tube stage. The spat at this time is used as the material, and its specifications are shell length: 750±50.86μm, shell height: 660±45.32μm; Juvenile oysters: After about 45 days of intermediate cultivation, the juvenile oysters (2mm) can be taken out of the pond and released into the sea. The juvenile oysters at this time are used as materials, with the specifications of shell length: 2.15±0.07cm, shell height: 2.04±0.03cm.

3. The method for studying the high temperature and low salt tolerance of Chinese taro at different developmental stages according to claim 1, characterized in that: The step S2 is specifically as follows: S2.

1. Under acute temperature rise conditions, five temperature gradients were set at 26°C (control group), 28°C, 30°C, 32°C, and 34°C, and three parallel experiments were set for each gradient. The experiment was carried out in a beaker or a plastic bucket. Then seawater was added. The beaker or plastic bucket was temperature-controlled in a water bath or directly heated using an automatic temperature control heating rod. The larvae density was 3 / mL, the juvenile density was 1 / mL, and the juvenile density was 40 / L. The salinity was maintained at 27-28 and the pH was 7.9-8.

1. The three developmental stages of the larvae, juveniles, and juveniles of the larvae of the larvae were respectively carried out as described above. S2.

2. Under chronic warming conditions, start from the temporary culture temperature (26°C), increase at a gradual rate of 1°C / d to 34°C in a beaker or a plastic bucket. Add seawater to the beaker or plastic bucket, and use an automatic temperature control heating rod to control the temperature of the beaker or plastic bucket in a water bath or directly heat it. Then put in the Chinese tongue with a larval density of 3 / mL, a juvenile density of 1 / mL, and a juvenile density of 40 / L. Maintain the salinity at 27-28 and the pH at 7.9-8.

1. Experiments were carried out in the three developmental stages of Chinese tongue larvae, juveniles, and juveniles as described above.

4. The method for studying the high temperature and low salt tolerance of Chinese taro at different developmental stages according to claim 1, characterized in that: The step S3 is specifically as follows: S3.

1. Starting from the temporary culture salinity (28‰), the salinity was gradually reduced at a rate of 2‰ / d until it reached 12‰. The experiment was set up in three parallels and was carried out in one of the beakers or plastic buckets. Seawater was added to the beakers or plastic buckets to maintain the water temperature at 25-26℃ and the pH at 7.9-8.

1. The larvae density was 3 / mL, the spat density was 1 / mL, and the juvenile density was 40 / L. The experiments were carried out in the three developmental stages of the larvae, juveniles, and juveniles of the Chinese tongue respectively according to the above. S3.2, then calculate the survival rates of the three developmental stages of the larvae, juveniles and young oysters under chronic low-salinity conditions; S3.

3. Data were expressed as "mean ± standard deviation (X ± SD)". The measurement indicators were processed using Excel 2019 software. SPSS22.0 software was used to perform one-way analysis of variance on the data. The Duncan's multiple comparison method was used to test the significance of differences between different treatments.

5. The method for studying the high temperature and low salt tolerance of Chinese taro at different developmental stages according to claim 4, characterized in that: In step S3.1, when the three developmental stages of the larvae, juveniles and young shellfish were studied under chronic low-salinity conditions, the algae density in the water body was maintained at (1-2)×10 4 cell / mL、(2-3)×10 4 cel l / mL and (5-10)×10 4 cell / mL.

6. The method for studying the high temperature and low salt tolerance of Chinese taro at different developmental stages according to claim 1, characterized in that: The calculation formula of step S3.2 is as follows: Actual survival rate formula: SR = N t / N0×100% Where, N0 is the initial density / number of Xishi tongue, N t is the terminal shishi tongue density / number; Relative survival rate formula: P = SR / SR' control group × 100% In the formula, SR is the actual survival rate, and SR' control group is the survival rate of the corresponding control group; The half-lethal temperature formula is: LT 50 =T1+[(P1-50%) / (P1-P2)]×(T2-T1) Where: T1 and T2 are the high and low end temperatures where the survival rate is close to 50%; P1 and P2 are the corresponding relative survival rates; The half-lethal salinity formula is: LS 50 =S1+[(P1-50%) / (P1-P2)]×(S2-S1) Where: S1 and S2 are the high and low end salinities with a survival rate close to 50%; P1 and P2 are the corresponding relative survival rates.

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