Crassostrea sikamea living body identification method based on salinity preference

Abstract

The invention discloses a method for identifying living crassostrea sikamea based on salinity preference, which comprises the following steps: collecting a plurality of crassostrea sikamea from a sea area in which wild crassostrea sikamea and wild Fujian crassostrea sikamea coexist, temporarily culturing the crassostrea sikamea and the wild Fujian crassostrea sikamea in a water body for 3 days, gradually reducing the salinity of the water body from 25 days to 10 days during the temporary culturing period, continuously inflating the water body during the period, changing 50% of water every day and feeding spirulina powder once; keeping the salinity of the water body at 10 + / -0.5 for 3 weeks, continuously inflating the water body during the period, changing water by 50% every day, feeding spirulina powder once, counting the number of dead individuals once every day, and timely taking out the dead individuals; the obtained survival oysters are cultured in normal mariculture and used for improved variety breeding work of Crassostrea sikamea. The Crassostrea sikamea living body identification method based on salinity preference is simple and efficient, the identified and screened Crassostrea sikamea is still a living body, a living body material is provided for purebred basic population construction, biological experiments and other purposes, and subsequent physiological experiments, breeding and the like are not affected.

Description

Technical Field

[0001] The invention relates to the field of identification, protection and application of aquatic germplasm resources, and in particular to a method for identifying live Kumamoto oysters (Crassostrea sikamea) based on salinity preference. Background Art

[0002] Oysters, commonly known as oyster roe and raw oysters, are widely distributed in all seas except the frigid zone. Guangdong, Guangxi, Fujian, Zhejiang, Shandong, Liaoning and other provinces in my country are the main oyster farming provinces, with an annual output of more than 6 million tons, accounting for about 1 / 4 of the marine aquaculture output, and have important economic value. The coastal areas of Zhejiang, Fujian, Guangdong and Guangxi are very rich in Kumamoto oyster resources. Xiangshan Port in Zhejiang is the core area of ​​natural distribution of Kumamoto oysters. The coastal areas of Zhejiang have become the largest area for Kumamoto oyster farming in the world. Due to its good taste and beautiful shell shape, Kumamoto oysters have gradually become a high-end oyster variety in the world.

[0003] Since the shell shape of oysters is greatly affected by the environment and individual differences are significant, it is impossible to effectively identify oysters based on their shell shape. In many sea areas in southern my country, Kumamoto oysters and Fujian oysters are distributed in the same area. In addition, under artificial breeding conditions, Kumamoto oysters can be fertilized unidirectionally with Fujian oysters to produce offspring. This is not conducive to the selection of Kumamoto oysters and the enhancement of the brand added value of Kumamoto oysters. In addition, in recent years, the oyster farming industry has developed rapidly, and the migration of oyster seedlings to other places for farming has become more and more frequent, which has had an impact on the wild oyster resources in various sea areas.

[0004] Accurate identification of species is the basis for conducting biological research and protecting germplasm resources. The methods of species identification involve multiple levels, mainly including the following methods: 1) Morphological methods: observing the external morphology, internal structure or cell morphology of organisms, which may involve measuring the length, shape, color, texture and other characteristics of the body parts of organisms; 2) Molecular methods: including DNA barcoding and molecular sequencing, among which DNA barcoding uses the sequence of specific gene fragments to identify species, and molecular sequencing identifies species by sequencing specific genomes or gene fragments; 3) Ecological characteristic methods: studying the ecological habits, habitat preferences, geographical distribution and other characteristics of species. This method can be identified by observing the behavior and environmental selection of organisms in natural environments; 4) Chemical analysis methods: using chemical methods to analyze chemical substances in organisms, such as metabolites, hormones or specific biomarkers. Species identification usually requires the combined use of multiple methods, especially for complex or similar species. For example, combining morphological characteristics and molecular methods can improve the accuracy and reliability of identification.

[0005] At present, the molecular methods used for oyster species identification include: PCR-RFLP identification, mitochondrial COI gene sequencing, multiple specific PCR, etc. However, these methods require DNA extraction and PCR identification, which require certain experimental conditions, and the oysters are easily damaged or die after identification, which is not conducive to subsequent oyster physiological experiments and reproduction. In addition, traditional methods rely on morphological and anatomical means. Due to the characteristics of oysters such as wide habitat and high phenotypic plasticity, and the influence of identification experience of identification personnel, this subjective method has the possibility of misjudgment, so it is impossible to effectively identify oysters. Therefore, it is very important to establish a live identification method for Kumamoto oysters and apply it to Kumamoto oyster breeding. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a simple and efficient method for identifying live Kumamoto oysters based on salinity preference in view of the deficiencies in the prior art. The identification method is based on the different tolerance of Kumamoto oysters and Fujian oysters to low salinity, and can play an important role in the protection of Kumamoto oyster germplasm resources and the large-scale rapid identification in the production and breeding process. The advantage of the identification method is that the Kumamoto oysters identified and screened are still alive, which provides living materials for the construction of purebred basic populations, biological experiments, etc., and does not affect subsequent physiological experiments and reproduction.

[0007] The inventors studied the tolerance of Kumamoto oysters and Fujian oysters under low-salinity conditions and found that salinity is an important factor affecting the growth of oysters. If subjected to salinity stress, oysters need to consume a large amount of energy to maintain their own osmotic pressure balance. Salinity can directly affect the physiological metabolism of oysters and thus affect the survival and growth of oysters, and different varieties of oysters have different adaptability to salinity. Kumamoto oysters are distributed in large quantities in the intertidal zone of the estuary, while Fujian oysters are almost not distributed in the low-salinity area of ​​the estuary. When Kumamoto oysters and Fujian oysters live in water bodies with low salinity, their own physiological regulation ability is weakened, and even death occurs, while Kumamoto oysters have better adaptability to low-salinity conditions. Therefore, the present invention proposes a method for identifying live Kumamoto oysters based on salinity preference. The identification method utilizes the difference in tolerance between Kumamoto oysters and Fujian oysters under specific low-salinity conditions, and is a simple and efficient method for identifying live Kumamoto oysters.

[0008] The technical solution adopted by the present invention to solve the above technical problems is: a method for identifying live Kumamoto oysters based on salinity preference, comprising the following steps:

[0009] (1) A number of oysters were collected from a sea area where wild Kumamoto oysters and wild Fujian oysters coexisted, and were temporarily cultured in water for 3 days. During the culture period, the salinity of the water was gradually reduced from 25 on the first day of culture to 10 on the third day of culture. During this period, the water was continuously aerated, the water volume was changed by 50% every day, and Spirulina powder filtered through a 300-mesh silk sieve was fed once;

[0010] (2) The salinity of the water body was maintained at 10±0.5 for 3 weeks, during which time the water body was continuously aerated, 50% of the water was changed every day, and Spirulina powder filtered through a 300-mesh silk sieve was fed once. The number of dead individuals was counted once a day, and the dead individuals were removed in time to prevent water quality corruption. Because the tolerance of Fujian oysters to low-salinity conditions was poor, all of them died in water bodies with a salinity of 10±0.5 for 3 weeks, while the tolerance of Kumamoto oysters to low-salinity conditions was strong, and the survival rate in water bodies with a salinity of 10±0.5 for 3 weeks was above 80%;

[0011] (3) The surviving oysters obtained after step (2) are cultured in normal seawater for use in the breeding of Kumamoto oysters.

[0012] Preferably, in step (1), the salinity of the water body on the second day of temporary storage is 18.

[0013] Preferably, in step (2), the method for determining the individual's death characteristics is as follows: if the individual's double valves are open and cannot close by themselves, and there is no response when stimulated with tweezers, the individual is determined to be dead.

[0014] Preferably, in step (3), the surviving oysters are moved to a pilot plant and cultured using normal seawater.

[0015] Compared with the prior art, the present invention has the following advantages: Based on the different tolerance of Kumamoto oysters and Fujian oysters to low salinity, the present invention takes advantage of the fact that Kumamoto oysters can tolerate low salinity better, and establishes a low-cost, simple and efficient method for identifying live Kumamoto oysters, which can play an important role in the protection of Kumamoto oyster germplasm resources and the large-scale rapid identification of production and breeding processes. Compared with traditional morphological methods, the identification method of the present invention has higher accuracy and is not affected by the identification experience of identification personnel; compared with conventional DNA molecular identification methods, the advantage of the identification method of the present invention is that the Kumamoto oysters identified and screened are still alive, providing living materials for the construction of purebred basic populations, biological experiments, etc., without affecting subsequent physiological experiments and reproduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a morphological comparison picture of Kumamoto oyster and Fujian oyster;

[0017] Figure 2 The electrophoresis diagram of DNA molecule identification of Kumamoto oyster and Fujian oyster in some embodiments;

[0018] Figure 3 These are the survival curves of Kumamoto oysters and Fujian oysters under the condition of water salinity of 10. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below with reference to the accompanying drawings.

[0020] Figure 1 This is a morphological comparison of the Kumamoto oyster and the Fujian oyster. Figure 1 (a) in the middle is Kumamoto oyster. Figure 1 (b) is a Fujian oyster, which cannot be distinguished by the appearance of the shell. The identification method of the present invention can be used to distinguish and screen the Kumamoto oyster from the Fujian oyster, and the method is as follows:

[0021] Embodiment: About 800 oysters were collected from the Xiangshan Port sea area in Zhejiang Province. After removing the attachments on the surface of the oyster shells, they were temporarily cultured in a laboratory seawater aquaculture system for 3 days. During the temporary culture period, the salinity of the water body was gradually reduced from 25 on the first day of temporary culture to 18 on the second day of temporary culture and 10 on the third day of temporary culture. During this period, the water body was continuously aerated, the water volume was changed by 50% every day, and spirulina powder filtered through a 300-mesh silk sieve was fed once. After the temporary culture, healthy and undamaged oyster individuals were selected, and the oysters were divided into two groups, suspected Kumamoto oysters and Fujian oysters, by traditional methods based on morphology. The experimental design was that the salinity of 15 was the normal salinity group S15, and the salinity of 10 was the low salt group S10. Each group had two parallels, and each parallel had 33 individuals preliminarily determined to be Fujian oysters and 100 individuals preliminarily determined to be Kumamoto oysters. During the experiment, the water body was continuously aerated, the water volume was changed by 50% every day, and Spirulina powder filtered through a 300-mesh silk sieve was fed once. The number of dead individuals was counted once a day, and the dead individuals were removed in time to prevent water corruption. The method for judging the characteristics of individual death is as follows: if the bivalve of the individual is opened and cannot close by itself, and there is no response when stimulated with tweezers, the individual is judged to be a dead individual. The adductor muscle of the dead oyster was taken, placed in a cryopreservation tube, quickly frozen with liquid nitrogen, and stored in a -80°C refrigerator. The accuracy of the method of the present invention was verified by DNA identification. After the experiment, 30 oysters were randomly selected from the surviving oysters, and the distribution of Kumamoto oysters and Fujian oysters in the surviving oysters was also verified by DNA methods.

[0022] DNA method verification: DNA was extracted from the retained adductor muscle, and the obtained DNA was amplified by multiple PCR using COI primers. The PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2. The primer sequences are:

[0023] COⅠ-AllF 5'-GGTCAACAAATCATAAAGATATTGG-3';

[0024] COⅠ-AllR 5'-TAAACTTCAGGGTGACCAAAAAATCA-3';

[0025] COⅠ-AN 5'-AGTTACCAAACCCCCCAATTATCAGG-3';

[0026] COⅠ-SI 5'-AAGTAACCTTAATAGATCAGGGAACC-3'.

[0027] Table 1 PCR reaction system

[0028]

[0029] Table 2 PCR reaction conditions

[0030]

[0031] Note: The PCR reaction consists of 35 cycles from annealing to extension.

[0032] The PCR products were subjected to 1% agarose gel electrophoresis to identify the oyster species. Figure 2 The electrophoresis diagram of DNA molecule identification of Kumamoto oyster and Fujian oyster in some embodiments is shown in FIG. Figure 2 Except for 3-4, 3-7, 3-21, 3-22, and 3-24, which are Kumamoto oysters, the rest are Fujian oysters. Figure 2 As shown, the Kumamoto oyster has a common band at around 700 bp and a Kumamoto oyster-specific band at around 600 bp, and the Fujian oyster has a common band at around 700 bp and a 200 bp-specific band.

[0033] After 3 weeks of stress, 30 oysters were randomly selected from the remaining surviving oysters and their species were identified using molecular methods. The species distribution of the surviving oysters is shown in Table 3.

[0034] Table 3 Distribution of surviving oyster species

[0035]

[0036] As shown in Table 3, under the condition of salinity 15, the proportion of Fujian oysters in the two groups of surviving oysters was 30% and 10%, respectively, while under the condition of salinity 10, the proportion of Fujian oysters in the two groups of surviving oysters was 0%. It can be inferred that after 3 weeks of treatment with a water salinity of 10, all Fujian oysters have died, and the remaining surviving oysters are all Kumamoto oysters.

[0037] Count the number of Kumamoto oysters and Fujian oysters among the dead oysters, and draw the survival curves of the two oysters under the condition of water salinity of 10, such as Figure 3 As shown. It was found that there was a significant difference in the survival rate of Kumamoto oysters and Fujian oysters. The mortality rate of both oysters did not increase significantly in the first 7 days, but as the stress time increased, the mortality rate of both oysters increased significantly, among which the mortality rate of Fujian oysters was much higher than that of Kumamoto oysters. After 21 days, the survival rate of Kumamoto oysters exceeded 80%, and all the surviving oysters were Kumamoto oysters, which can be used in subsequent physiological experiments and reproduction.

Claims

1. A method for identifying live Kumamoto oysters based on salinity preference, characterized in that: The following steps are involved: (1) A number of oysters were collected from a sea area where wild Kumamoto oysters and wild Fujian oysters coexisted, and were temporarily cultured in water for 3 days. During the culture period, the salinity of the water was gradually reduced from 25 on the first day of culture to 10 on the third day of culture. During this period, the water was continuously aerated, the water volume was changed by 50% every day, and Spirulina powder filtered through a 300-mesh silk sieve was fed once; (2) The salinity of the water body was maintained at 10±0.5 for 3 weeks, during which time the water body was continuously aerated, the water volume was changed by 50% every day, and Spirulina powder filtered through a 300-mesh silk sieve was fed once, and the number of dead individuals was counted once a day and the dead individuals were removed in time. Since the tolerance of Fujian oysters to low-salinity conditions was poor, all of them died in water bodies with a salinity of 10±0.5 for 3 weeks, while the tolerance of Kumamoto oysters to low-salinity conditions was strong, and the survival rate in water bodies with a salinity of 10±0.5 for 3 weeks was above 80%; (3) The surviving oysters obtained after step (2) are cultured in normal seawater for use in the breeding of Kumamoto oysters.

2. A method for identifying live Kumamoto oysters based on salinity preference according to claim 1, characterized in that: In step (1), the salinity of the water body on the second day of temporary storage is 18.

3. The method for identifying live Kumamoto oysters based on salinity preference according to claim 1, characterized in that: In step (2), the method for determining the individual's death characteristics is as follows: if the individual's double valves are open and cannot close by themselves, and if there is no response when stimulated with tweezers, the individual is determined to be dead.

4. The method for identifying live Kumamoto oysters based on salinity preference according to claim 1, characterized in that: In step (3), the surviving oysters are moved to a pilot plant and cultured using normal seawater.

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