A sample pretreatment method for determining stable isotope composition of reef-building coral host and symbiotic zooxanthellae

Abstract

This invention belongs to the field of stable isotope determination technology. It provides a sample pretreatment method for determining the stable isotopic composition of host corals and their symbiotic zooxanthellae. Addressing the problems of incomplete separation of host and symbiotic zooxanthellae and interference from impurities such as coral skeletons in sample pretreatment, this invention systematically optimizes the host-zooxanthellae separation process based on air gun peeling, graded silk sieve filtration, and centrifugal washing, and introduces microscopic examination for purity control. By comparing the effects of phosphate-buffered saline (PBS) and ultrapure water as rinsing media, ultrapure water is recommended as the standard rinsing medium. This method is standardized, reproducible, and provides a reliable technical foundation for coral nutritional ecology research, especially suitable for analyzing the carbon and nitrogen distribution and sources within symbiotic systems.

Description

Technical Field

[0001] This invention relates to the field of stable isotope determination technology, and in particular to a sample pretreatment method for determining the stable isotopic composition of reef-building coral hosts and symbiotic zooxanthellae. Background Technology

[0002] Reef-building corals, as the framework builders of coral reef ecosystems, are "holistic organisms" composed of their cnidarian hosts and endosymbiotic dinoflagellates and zooxanthellae. Their nutritional strategies exhibit both autotrophic and heterotrophic characteristics: on the one hand, they rely on zooxanthellae photosynthesis to fix organic carbon; on the other hand, they obtain exogenous energy and nutrients through the host's ingestion of dissolved organic matter, suspended particles, and plankton. In nutrient-poor tropical waters, this diversified nutritional model is a crucial foundation for maintaining high coral reef productivity and ecological functions. Stable isotope techniques (especially carbon and nitrogen isotope composition) are essential. and As an effective natural tracing method, it has been widely used to reveal the energy sources, nutritional status, and responses of corals to environmental changes.

[0003] In recent years, with the development of research methods, separating and determining the stable isotopic composition of the host and zooxanthellae has become a key means to deeply analyze the nutrient allocation pattern and symbiotic interaction mechanism of corals. Studies have shown that there are systematic differences in δ¹³C between the host and zooxanthellae, and that the composition exhibits plasticity with changes in light, water depth, and nutrient conditions; isotope tracing experiments (such as...) can be used to further analyze the composition of the host and zooxanthellae. , The use of markers can further quantitatively assess the energy budget and nutritional strategy adjustments of corals under different environmental conditions. Therefore, integrating stable isotope analysis with host-zooxanthellae separation technology is of great significance for systematically revealing the nutritional dynamics of corals and their adaptation mechanisms to environmental stress.

[0004] However, at the experimental level, there is still a lack of unified and reproducible standard procedures for coral sample pretreatment and host-zooxanthellae separation. Furthermore, coral samples often contain skeletal debris, mucus, and salts, which can easily interfere with organic carbon and nitrogen content and isotope measurements if not properly treated. Therefore, establishing a standardized, quality-controlled, and reproducible sample pretreatment method is of great significance for advancing research on coral stable isotope ecology. Summary of the Invention

[0005] The purpose of this invention is to provide a sample pretreatment method for determining the stable isotopic composition of reef-building coral hosts and symbiotic zooxanthellae, addressing the shortcomings of existing technologies.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a sample pretreatment method for determining the stable isotopic composition of reef-building coral hosts and symbiotic zooxanthellae, comprising the following steps: 1) Immerse the coral sample in the rinsing medium and rinse with an air gun to detach the host coral tissue and symbiotic zooxanthellae from the skeleton, forming a mixed suspension; 2) The mixed suspension was passed through two stages of sieve filtration to remove coral skeleton fragments and larger particulate impurities, and the filtrate was obtained. 3) The filtrate is centrifuged to obtain zooxanthellae precipitate and supernatant, wherein the supernatant is collected and preserved as coral host components; 4) Add washing medium to the zooxanthellae precipitate for resuspension, centrifuge under centrifugation conditions and discard the supernatant. Repeat the washing cycle of resuspension, centrifugation and discarding supernatant to remove residual host tissue fragments and salts, and detect the separation purity by microscopy to obtain the zooxanthellae component. 5) The zooxanthellae component and the coral host component were subjected to freeze-drying, freeze-drying, grinding, and packaging processes for stable isotope composition determination. The stable isotope composition was a carbon stable isotope composition. and nitrogen stable isotopes .

[0007] Preferably, the coral sample in step 1) is a healthy reef-building coral collected by scuba diving at a depth of 3-5m. After collection, it is rinsed with seawater to remove surface sediments and visible large epiphytes, then placed in a sealed container, protected from light, and refrigerated before being transported back to the laboratory. Cryopreservation or acclimatization in an experimental coral tank for 6-8 days.

[0008] Preferably, the rinsing medium in step 1) is ultrapure water pre-cooled at 3~5℃; The pressure of the air gun flushing is 0.1~0.2MPa.

[0009] Preferably, the two-stage sieving and filtration in step 2) is carried out using a sieve with a pore size of 180~220μm and a sieve with a pore size of 40~60μm, respectively.

[0010] Preferably, the centrifugation temperature in step 3) is 3~5℃, the centrifugation force is 4000~6000×g, and the centrifugation time is 8~12min.

[0011] Preferably, the washing medium in step 4) is ultrapure water pre-cooled at 3~5℃; The centrifugation conditions are: temperature 3~5℃, centrifugal force 4000~6000×g, and centrifugation time 8~12min.

[0012] Preferably, in step 4), the separation purity is determined by microscopic examination, with the proportion of non-zooxanthellae particles no longer decreasing significantly, and the zooxanthellae cells remaining intact and the background clean.

[0013] Preferably, the freezing temperature in step 5) is The freezing time is 20-26 hours; The freeze-drying temperature is: , freeze-dried to constant weight.

[0014] Preferably, the grinding in step 5) is performed using a cryosurgical grinder, with a grinding temperature of 0~4℃, a frequency of 20~30Hz, a single grinding time of 15~20s, a grinding interruption time of 2~4s, and a grinding number of 5~10 times.

[0015] Preferably, during the encapsulation process in step 5), a balance of 1 / 1 million is used to weigh the sample, with the coral host component weighing 550-600 μg and the zooxanthellae component weighing 190-210 μg.

[0016] The beneficial effects of this invention include the following: 1) This invention reduces the impact of coral skeletons on the results by establishing a repeatable operation procedure based on air gun peeling, silk sieve filtration and centrifugal separation.

[0017] 2) This invention achieves quality control of the separation process by introducing microscopic examination and quantitative purity criteria.

[0018] 3) This invention compares the effects of different rinsing media (such as PBS and ultrapure water) on coral hosts and zooxanthellae. and The systemic impact, while optimizing the injection volume to enable simultaneous analysis and As a result, data reliability is improved.

[0019] 4) The method described in this invention is standardized and reproducible, providing a reliable technical foundation for coral nutritional ecology research, and is especially suitable for analyzing the carbon and nitrogen distribution and sources within symbiotic systems. Attached Figure Description

[0020] Figure 1 The correlation diagram shows the relationship between the amount of sample injected into the coral host and the carbon and nitrogen signals. In the diagram, A represents the relationship between the nitrogen signal and the amount of sample injected, and B represents the relationship between the carbon signal and the amount of sample injected. Figure 2 The correlation diagram shows the relationship between the amount of zooxanthellae injected and the carbon and nitrogen signals. In the diagram, A represents the relationship between the nitrogen signal and the amount of injected sample, and B represents the relationship between the carbon signal and the amount of injected sample. Figure 3 Carbon stable isotopes The purification effect diagram; Figure 4 Nitrogen stable isotopes The purification effect diagram; Figure 5 The results of stable isotope analysis at different field sites are shown in the figure. (a) is the isotope niche distribution at different sites, and (b) is the nitrogen isotope difference at different sites. Figure 6 A comparison diagram showing the distribution of stable isotopes under different flushing media conditions. Detailed Implementation

[0021] This invention provides a sample pretreatment method for determining the stable isotopic composition of reef-building coral hosts and symbiotic zooxanthellae, comprising the following steps: 1) Immerse the coral sample in the rinsing medium and rinse with an air gun to detach the host coral tissue and symbiotic zooxanthellae from the skeleton, forming a mixed suspension; 2) The mixed suspension was passed through two stages of sieve filtration to remove coral skeleton fragments and larger particulate impurities, and the filtrate was obtained. 3) The filtrate is centrifuged to obtain zooxanthellae precipitate and supernatant, wherein the supernatant is collected and preserved as coral host components; 4) Add washing medium to the zooxanthellae precipitate for resuspension, centrifuge under centrifugation conditions and discard the supernatant. Repeat the washing cycle of resuspension, centrifugation and discarding supernatant to remove residual host tissue fragments and salts, and detect the separation purity by microscopy to obtain the zooxanthellae component. 5) The zooxanthellae component and the coral host component were subjected to freeze-drying, freeze-drying, grinding, and packaging processes for stable isotope composition determination. The stable isotope composition was a carbon stable isotope composition. and nitrogen stable isotopes .

[0022] In this invention, the coral sample in step 1) is preferably a healthy reef-building coral collected by scuba diving. The collection depth is preferably 3-5m, more preferably 3.5-4.5m, and even more preferably 4m. After collection, it is preferably rinsed with seawater to remove surface sediments and visible large epiphytes, placed in a sealed container, protected from light, and refrigerated before being transported back to the laboratory. Freezing is preferred, and the optimal freezing temperature is [not specified]. Further optimized Alternatively, they can be cultured and acclimatized in an experimental coral tank for 6-8 days, with 7 days being the preferred time.

[0023] In this invention, the rinsing medium in step 1) is preferably ultrapure water pre-cooled at 3~5°C, and more preferably ultrapure water pre-cooled at 4°C, which can avoid the introduction of inorganic salts and buffer components, diluting the content of organic carbon and nitrogen, and causing the difference in stable isotope ratios within the group to increase.

[0024] In this invention, the pressure of the air gun rinsing in step 1) is preferably 0.1~0.2MPa, more preferably 0.14~0.16MPa, and even more preferably 0.15MPa. This pressure can ensure that the coral tissue is rinsed off while minimizing the amount of bone washed off.

[0025] In this invention, the two-stage sieving and filtration in step 2) is preferably performed by sequentially using pore sizes of 180~220μm. The process involves using a sieve with a pore size of 40-60 μm, and more preferably, a sieve with a pore size of 200 μm and a sieve with a pore size of 50 μm, in sequence, to remove the coral skeleton and coral mucus aggregates washed off in step 1).

[0026] In this invention, the centrifugation temperature in step 3) is preferably 3~5℃, more preferably 4℃, the centrifugation force is preferably 4000~6000×g, more preferably 4500~5500×g, more preferably 5000×g, and the centrifugation time is preferably 8~12min, more preferably 9~11min, more preferably 10min.

[0027] In this invention, the washing medium in step 4) is preferably ultrapure water pre-cooled at 3~5°C, and more preferably ultrapure water pre-cooled at 4°C; The preferred centrifugation conditions are: temperature of 3~5℃, centrifugal force of 4000~6000×g, and centrifugation time of 8~12min; more preferably, temperature of 4℃, centrifugal force of 5000×g, and centrifugation time of 10min.

[0028] In this invention, step 4) involves detecting the separation purity using a microscope. Preferably, the standard for determining whether the separation purity meets the criteria is that the proportion of non-zooxanthellae particles no longer decreases significantly, and that the zooxanthellae cells are intact and the background is clean.

[0029] In this invention, the freezing temperature in step 5) is preferably... Further optimized More preferably The freezing time is preferably 20-26 hours, more preferably 22-24 hours, and even more preferably 23 hours; The preferred temperature for freeze-drying is... Further optimized More preferably Preferably, it is freeze-dried to constant weight.

[0030] In this invention, the grinding in step 5) is preferably performed using a cryosurgical; the grinding temperature is preferably 0~4℃, more preferably 1~3℃, and even more preferably 2℃; the grinding frequency is preferably 20~30Hz, more preferably 24~26Hz, and even more preferably 25Hz; the grinding time for a single pass is preferably 15~20s, more preferably 16~18s, and even more preferably 17s; the grinding interruption time is preferably 2~4s, more preferably 3s; and the number of grinding passes is preferably 5~10, more preferably 6~8, and even more preferably 7.

[0031] In this invention, the grinding in step 5) mainly involves grinding the zooxanthellae components and coral host components to obtain a uniformly sized and well-mixed powdered sample of coral host and zooxanthellae, thereby reducing test deviation.

[0032] In this invention, during the encapsulation process in step 5), it is preferable to use a 1 / 1,000,000 balance for sample weighing. The preferred mass of the coral host component is 550-600 μg, more preferably 554-556 μg, and even more preferably 555 μg. The preferred mass of the zooxanthellae component is 190-210 μg, more preferably 195-205 μg, and even more preferably 200 μg. This weighing range ensures good response intensity for both N and C signals. With increasing sample volume, both the N and C signals of the zooxanthellae and coral host samples show a significant linear upward trend. The correlation between the coral host sample volume and the carbon and nitrogen signals is shown in the figure below. Figure 1 As shown, A represents the relationship between nitrogen signal and injection volume, and B represents the relationship between carbon signal and injection volume; the correlation graphs between zooxanthellae injection volume and carbon and nitrogen signals are shown in the figure. Figure 2 As shown, A represents the relationship between the nitrogen signal and the injection volume, and B represents the relationship between the carbon signal and the injection volume. Figure 1 and Figure 2 It was found that the sample amount of both coral host and zooxanthellae samples showed a good linear correlation with both carbon and nitrogen signals. Under low sample amounts (<300 μg), the nitrogen signal of some samples approached or fell below the instrument's detection limit, potentially affecting the stability of isotope measurements. Under high sample amounts (>900 μg), the carbon signal of zooxanthellae samples gradually approached the upper limit of the linear response, posing a potential risk of saturation. Considering the dual requirements of the N signal being above the detection limit and the C signal being below the saturation limit, the recommended sample amount for zooxanthellae samples was determined to be 190–210 μg, and for coral host samples, 550–600 μg. Within these ranges, the signal intensity is stable and within the instrument's linear response range.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1: This embodiment provides a method for determining the stable isotopic composition of reef-building coral hosts and symbiotic zooxanthellae. and The sample pretreatment method specifically includes the following steps: 1) On April 30, 2024, healthy reef-building coral samples were collected by scuba diving at a depth of 3-5 meters in the waters off Tongguling in Wenchang and Jiajing Island in Wanning. The collected coral species were staghorn cup coral and clustered helmet coral. After collection, the coral surface was gently rinsed with seawater to remove attached sediment and visible large epiphytes. The samples were then placed in sealed plastic bags, protected from light, and refrigerated before being transported back to the laboratory. Store frozen under the specified conditions for future use.

[0035] After the frozen coral samples were taken out, they were placed on an ice plate to thaw and then placed in a sterile self-sealing bag. Pre-cooled ultrapure water at 4°C was added as a rinsing medium to completely submerge the samples. The samples were then rinsed with air jets at a pressure of 0.2 MPa to detach the coral host tissues and symbiotic zooxanthellae from the skeleton, forming a mixed suspension.

[0036] 2) The resulting mixed suspension was subjected to a two-stage sieve filtration process, first through a sieve with a pore size of 200 μm. The coral skeleton fragments were initially filtered through a sieve to remove visible coral skeleton fragments; then, a second filtration was performed through a sieve with a pore size of 50 μm to further remove larger fragments of host tissue and aggregates, resulting in a filtrate.

[0037] 3) Place the above filtrate in a centrifuge tube and centrifuge at 5000×g for 10 min at 4℃ to obtain zooxanthellae precipitate and supernatant, wherein the supernatant is collected and stored as coral host component.

[0038] 4) Add pre-cooled ultrapure water (4°C) to the zooxanthellae precipitate as a washing medium for resuspending (calculated as 2 mL ultrapure water per mL zooxanthellae precipitate). Centrifuge at 4°C and 5000×g for 10 min, then discard the supernatant. Repeat the resuspending, centrifugation, and supernatant discarding cycle to remove residual host tissue fragments and salts. Continue until microscopic observation shows that the proportion of non-zooxanthellae particles no longer decreases significantly, and the zooxanthellae cells are morphologically intact with a clean background. At this point, the separation purity is considered satisfactory, and the purified zooxanthellae fraction is obtained. Carbon stable isotopes The purification effect diagram is as follows Figure 3 As shown, nitrogen stable isotopes The purification effect diagram is as follows Figure 4 As shown. By Figure 3 and Figure 4It can be seen that: after treatment by the method of the present invention, the carbon stable isotopes of the zooxanthellae components are... and nitrogen stable isotopes The reduced fluctuations and more concentrated data in the measurement results indicate that the washing and separation operations effectively removed interference from host residues and salts, achieving sample purification. At the same time, the minimal changes in host components further demonstrate that this method can effectively separate the host from zooxanthellae.

[0039] 5) After collecting the isolated zooxanthellae and coral host components separately, they were then... Freeze for 24 hours under the specified conditions, then freeze-dry under a vacuum of 20 Pa until constant weight. The freeze-drying temperature is [temperature missing]. The freeze-dried samples were ground using a cryo-tissue homogenizer at a temperature of 3°C and a frequency of 25 Hz. Each grinding cycle lasted 16 seconds, with a 3-second interval between cycles, and the process was repeated 8 times to obtain a uniform powder sample.

[0040] Using a 1 / 1,000,000 balance, 190–210 μg of zooxanthellae samples and 550–600 μg of coral host samples were weighed and placed in tin cups for stable isotope determination.

[0041] The above samples were analyzed using a stable isotope ratio mass spectrometer. and Measurements were performed. The results showed that the coral host at Wenchang Station... The mean value was 8.89±0.27‰ (range 8.64~9.24‰), and the mean value of zooxanthellae was 8.21±0.42‰ (range 7.55~8.69‰); coral hosts at Wanning Station The mean value was 5.81±0.69‰ (range 5.01~6.74‰), and the mean value of zooxanthellae was 6.25±1.43‰ (range 4.27~7.90‰). Wenchang Station as a whole... The concentration of inorganic nitrogen at the Wanning station was approximately 2.4–3.1‰ higher than that at the Wanning station, indicating a significant difference in the source or nutrient environment of inorganic nitrogen between the two locations. Regarding carbon isotopes, the host at the Wanning station... The mean is Zooxanthellae are Zooxanthin algae were significantly enriched. The carbon content was approximately 1.8‰ higher than that of the host, suggesting that the host still has a certain heterotrophic carbon source; the host at Wenchang Station The mean is Zooxanthellae are The difference between the two is only about 0.2‰, reflecting that the carbon source of the host at this site is highly dependent on symbiotic photosynthesis.

[0042] Figure 1 shows the results of stable isotope analysis at different field sites. Figure 5As shown, (a) is an isotopic niche distribution map of different sites, and (b) is a nitrogen isotope difference map of different sites. Figure 5 It can be seen that: between different sites and The significant differences indicate that the nitrogen source characteristics and nutritional patterns of corals in different sea areas exhibit substantial spatial differentiation, demonstrating that the method of this invention can effectively distinguish the stable isotopic composition of coral hosts and zooxanthellae, and that the measurement results are stable and reliable.

[0043] Example 2: This embodiment provides a method for determining the stable isotopic composition of reef-building coral hosts and symbiotic zooxanthellae. and The sample pretreatment method was described, and the effects of different rinsing media on the results of stable isotope determination were compared. The specific steps included: 1) On April 30, 2025, healthy reef-building coral samples were collected from the Wenchang coral reef area at a depth of 3-5m using scuba diving. The collected coral species were staghorn cup coral and clustered helmet coral. The collected coral fragments were gently rinsed with seawater to remove surface sediment and visible large epiphytes. The samples were then placed in sealed plastic bags, protected from light, and refrigerated before being transported back to the laboratory for 7 days of cultivation and acclimatization in a laboratory coral tank.

[0044] After domestication, the coral samples were placed in sterile self-sealing bags and rinsed with 4°C pre-cooled ultrapure water (pure water group) and PBS buffer (PBS group) as rinsing media to ensure complete immersion. The samples were then rinsed with air jets at a pressure of 0.1 MPa to detach the coral host tissue and symbiotic zooxanthellae from the skeleton, forming a mixed suspension.

[0045] 2) The obtained mixed suspension was subjected to two-stage sieve filtration. First, it was initially filtered through a sieve with a pore size of 200 μm to remove visible coral skeleton fragments. Then, it was filtered a second time through a sieve with a pore size of 50 μm to further remove larger host tissue fragments and aggregates, resulting in filtrate.

[0046] 3) Place the above filtrate in a centrifuge tube and centrifuge at 6000×g for 12 min at 3℃ to obtain zooxanthellae precipitate and supernatant, wherein the supernatant is collected and stored as coral host component.

[0047] 4) Add 4°C pre-cooled ultrapure water to the zooxanthellae precipitate as a washing medium for resuspending (calculated by adding 2 mL of ultrapure water to 1 mL of zooxanthellae precipitate). Centrifuge at 5°C and 4000×g for 8 min and discard the supernatant. Repeat the above washing cycle of resuspending, centrifuging and discarding supernatant to remove residual host tissue fragments and salts until the proportion of non-zooxanthellae particles no longer decreases significantly when observed under a microscope, and the zooxanthellae cells are intact and the background is clean. The separation purity is then determined to be up to standard, and the purified zooxanthellae component is obtained.

[0048] 5) After collecting the isolated zooxanthellae and coral host components separately, they were then... Freeze for 20 hours under the specified conditions, then freeze-dry under a vacuum of 50 Pa until constant weight. The freeze-drying temperature is [temperature missing]. The freeze-dried samples were ground using a cryo-tissue homogenizer at a temperature of 4°C, a frequency of 20 Hz, a single grinding time of 20 s, a grinding interruption time of 4 s, and a grinding count of 5 times to obtain a uniform powder sample.

[0049] Using a 1 / 1,000,000 balance, 190–210 μg of zooxanthellae samples and 550–600 μg of coral host samples were weighed and placed in tin cups for stable isotope determination.

[0050] 6) Stable isotope ratio mass spectrometry was used to determine the coral host components and zooxanthellae components in the pure water group and PBS group, respectively. and Values. The results showed that in the coral host component, the pure water group... The average value was 3.45 ± 0.06‰ (range 3.40 ~ 3.52‰). The average value is (scope ); PBS group The average value was 2.74 ± 1.08‰ (range 1.53 ~ 4.10‰). The average value is (scope ),in The overall concentration was slightly lower than normal (approximately 0.8‰), with significantly increased intra-group variability. Within the zooxanthellae group, the pure water group... The average value was 3.40 ± 0.13‰ (range 3.26 ~ 3.51‰). The average value is (scope PBS group The average value was 3.07 ± 1.01‰ (range 1.67 ~ 4.10‰). The average value is (scope Similarly, it also exhibits significant volatility.

[0051] The above results indicate that PBS treatment, by introducing inorganic salts and buffer components, dilutes the organic carbon and nitrogen content in the samples, leading to a significant increase in the intra-group variability of stable isotope ratios, thereby reducing the stability and reliability of the measurement results. A comparison of stable isotope distributions under different rinsing media conditions is shown in the figure below. Figure 6 As shown. By Figure 6 It can be seen that there are significant differences in the stable isotope distribution under different rinsing media conditions, with the PBS-treated group showing significantly greater fluctuations than the pure water group. In contrast, using ultrapure water as the rinsing and washing media yields more stable and reproducible results. and The results show that PBS is not suitable as a routine rinsing medium for coral stable isotope pretreatment without additional desalting. Ultrapure water is preferred for air gun rinsing and washing.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A sample pre-treatment method for determining the stable isotope composition of reef-building coral hosts and their symbiotic zooxanthellae, characterized in that, It includes the following steps: 1) Immerse the coral sample in the rinsing medium and rinse with an air gun to detach the host coral tissue and symbiotic zooxanthellae from the skeleton, forming a mixed suspension; 2) The mixed suspension was passed through two stages of sieve filtration to remove coral skeleton fragments and larger particulate impurities, and the filtrate was obtained. 3) The filtrate is centrifuged to obtain zooxanthellae precipitate and supernatant, wherein the supernatant is collected and preserved as coral host component; 4) Add washing medium to the zooxanthellae precipitate for resuspension, centrifuge under centrifugation conditions and discard the supernatant. Repeat the washing cycle of resuspension, centrifugation and discarding supernatant to remove residual host tissue fragments and salts, and detect the separation purity by microscopy to obtain the zooxanthellae component. 5) The zooxanthellae component and the coral host component are separately subjected to freezing, freeze-drying, grinding and encapsulation for stable isotope composition determination, the stable isotope composition being carbon stable isotope and nitrogen stable isotope .

2. The sample pretreatment method according to claim 1, characterized in that, Step 1) The coral sample is a healthy reef-building coral collected by artificial snorkeling, the collection water depth is 3-5 m, after collection, the surface attached sediments and visible epiphytes are removed by seawater washing, and the coral is packed into a sealed container, stored in the dark and cold, and transported back to the laboratory, and then frozen or cultured for 6-8 days in an experimental coral tank.

3. The sample preparation method according to claim 1 or 2, characterized in that, Step 1) The rinsing medium is ultrapure water pre-cooled at 3~5℃; The pressure of the air gun flushing is 0.1~0.2MPa.

4. The sample preparation method of claim 3, wherein, Step 2) The two-stage sieving and filtration process uses sieves with a pore size of 180~220μm and sieves with a pore size of 40~60μm, respectively.

5. The sample preparation method of claim 4, wherein, Step 3) The centrifugation temperature is 3~5℃, the centrifugal force is 4000~6000×g, and the centrifugation time is 8~12min.

6. The sample preparation method according to claim 4 or 5, characterized in that, Step 4) The washing medium is ultrapure water pre-cooled at 3~5℃; The centrifugation conditions are: temperature 3~5℃, centrifugal force 4000~6000×g, and centrifugation time 8~12min.

7. The sample preparation method of claim 6, wherein, Step 4) The separation purity is detected by microscope. The standard for judging whether the separation purity meets the standard is that the proportion of non-zooxanthellae particles no longer decreases significantly, and the zooxanthellae cells are intact and the background is clean.

8. The sample pretreatment method according to claim 7, characterized in that, Step 5) the temperature of said freezing is , freeze-dried to constant weight.

9. The sample preparation method according to claim 7 or 8, characterized in that, Step 5) The grinding is performed using a cryosurgical, with a grinding temperature of 0~4℃, a frequency of 20~30Hz, a single grinding time of 15~20s, a grinding interruption time of 2~4s, and a grinding number of 5~10 times.

10. The sample preparation method of claim 9, wherein, In step 5), during the encapsulation process, a balance of 1 / 1 million is used to weigh the samples. The mass of the coral host component is 550-600 μg, and the mass of the zooxanthellae component is 190-210 μg.

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