Albinized antler cup coral recovery method based on zooxanthellae inoculation

Abstract

The invention discloses an albino antler cup coral recovery method based on zooxanthellae inoculation, and belongs to the technical field of coral breeding. The method comprises the following steps: (1) firstly inoculating zooxanthellae into a culture medium, collecting zooxanthellae cells in an exponential growth phase, then transferring the zooxanthellae cells into seawater for nutrition starvation treatment, and finally centrifugally collecting the zooxanthellae cells and resuspending the zooxanthellae cells in the seawater to prepare a colonized zooxanthellae solution; and (2) carrying out light-dark period cycle culture on the albino coral, and periodically transferring the albino coral into a fixed-planting zooxanthellae solution for fixed planting in a light period to complete the recovery of the albino coral. The method is efficient, rapid and stable, field planting of albino coral by specific zooxanthellae can be rapidly completed based on co-incubation of zooxanthellae and coral, the fundamental problem that the symbiotic relationship between hermatypic coral and zooxanthellae in albino coral is broken is solved, rapid and stable recovery of albino coral is achieved, and the coral yield is increased. The method plays an important technical support for super coral breeding based on high-resistance zooxanthellae.

Description

Technical Field

[0001] The present invention relates to the technical field of coral cultivation, and particularly relates to a method for restoring bleached Acropora cervicornis based on zooxanthella inoculation. Background Art

[0002] Coral reef ecosystems are one of the important marine ecosystems in China. Especially in the South China Sea region, they are not only hotspots of biodiversity but also play an irreplaceable role in maintaining the marine ecological balance and fishery resources. However, in recent years, affected by global climate change, marine heatwaves have occurred frequently, leading to severe damage to the coral reef ecosystems in the South China Sea. The increase in seawater temperature caused by marine heatwaves directly induces large-scale bleaching events of reef-building corals. The essence of coral bleaching is the rupture of the symbiotic relationship between the host coral and zooxanthellae. As a single-celled alga, zooxanthellae live in the endodermal cells of corals and provide energy and nutrients for the host corals through photosynthesis. However, when the seawater temperature rises abnormally, the photosynthetic system of zooxanthellae will be damaged, generating excessive reactive oxygen species, resulting in damage to the coral host cells. To protect itself, the host coral will expel zooxanthellae, thus losing its main energy source, and ultimately leading to coral bleaching and even death.

[0003] Large-scale coral bleaching not only makes corals lose their colors but also severely weakens their survival ability and ecological functions. The bleached reef-building corals are more vulnerable to diseases, with a slower growth rate, a decline in reproductive ability, and may even lead to the collapse of the entire coral reef ecosystem. The degradation of coral reefs in the South China Sea region of China has had a significant impact on marine biodiversity and fishery resources, and at the same time threatens the livelihoods of coastal communities and the safety of the coastline. Facing this severe situation, it is urgent to strengthen the protection and management of coral reefs. Therefore, in addition to long-term measures such as reducing greenhouse gas emissions and controlling marine pollution, it is particularly urgent to develop a rapid and effective technology for restoring coral bleaching.

[0004] Currently, the commonly used means for restoring coral bleaching internationally mainly include coral transplantation, artificial reef construction, etc. Coral transplantation is to transplant healthy coral fragments to degraded areas to promote the recovery of coral communities. Artificial reef construction is to provide an attachment substrate for coral larvae by placing artificial structures to help rebuild coral populations. However, these means all have certain limitations. Although coral transplantation and artificial reef construction can improve the coral coverage rate in local areas in the short term, they are costly, and the transplanted corals may not be able to adapt to the new environmental conditions, resulting in limited recovery effects. Summary of the Invention

[0005] To solve the above technical problems, the object of the present invention is to provide a method for restoring bleached Acropora cytherea based on zooxanthella inoculation, so as to solve the problems of high cost of existing coral bleaching means, the transplanted corals may not be able to adapt to the new environmental conditions, and the limited restoration effect, etc.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: A method for restoring bleached Acropora cytherea based on zooxanthella inoculation, comprising the following steps: (1) Preparation of the zooxanthella solution for colonization: First, inoculate zooxanthellae into a culture medium, collect zooxanthella cells during the exponential growth phase, then transfer them into seawater for nutrient starvation treatment, and finally centrifuge to collect zooxanthella cells and resuspend them in seawater to obtain it; (2) Co-incubation of bleached corals and zooxanthellae: Carry out light-dark cycle culture on the bleached corals, and regularly transfer the bleached corals to the zooxanthella solution for colonization obtained in step (1) during the light period to complete the restoration of the bleached corals.

[0007] The beneficial effects of the present invention are as follows: The method of the present invention is efficient, rapid and stable. Based on the co-incubation of zooxanthellae and corals, it can quickly complete the colonization of specific zooxanthellae on bleached corals, solve the fundamental problem of the broken symbiotic relationship between reef-building corals and zooxanthellae in bleached corals, and thus achieve the purpose of quickly and stably restoring bleached corals.

[0008] Further, in step (1), the culture medium is f / 2 culture medium.

[0009] Further, the time of the nutrient starvation treatment in step (1) is 3 - 4 days.

[0010] The beneficial effect of adopting the above further technical solution is: By subjecting the zooxanthella cells to nutrient starvation treatment, the present invention enables them to consume the excess stored nutrients and stimulates their better transformation from the free state to the symbiotic state.

[0011] Further, in step (1), the concentration of zooxanthellae in the zooxanthella solution for colonization is 5×10 3 -5×10 4 cells / mL.

[0012] The beneficial effect of adopting the above further technical solution is: By optimizing the cell concentration in the zooxanthella concentrate, the present invention promotes the colonization efficiency of zooxanthellae and achieves an efficient and ideal colonization effect.

[0013] Further, the coral is Acropora cytherea.

[0014] Furthermore, the time for cyclic culture with light and dark periods in step (2) is 20 - 40 days, the light period is 10 - 14 h per day, and the dark period is 10 - 14 h per day.

[0015] Preferably, the time for cyclic culture with light and dark periods in step (2) is 30 days, the light period is 12 h per day, and the dark period is 12 h per day.

[0016] Furthermore, the colonization conditions in step (2) are as follows: transfer the bleached corals to the symbiotic zooxanthellae solution during the light period. After their tentacles naturally open, start timing for colonization for 20 - 40 min, and colonize 3 - 4 times a day.

[0017] Furthermore, feeding with Artemia nauplii is supplemented during colonization in step (2).

[0018] Furthermore, the light intensity during the light period in step (2) is 100 - 150 μE m -2 s -1 ; the culture parameters for both the light period and the dark period are: salinity 30 - 35 PSU, temperature 20 - 30 °C.

[0019] Preferably, the light intensity during the light period in step (2) is 120 μE m -2 s -1 ; the culture parameters for both the light period and the dark period are: salinity 33 PSU, temperature 25 °C.

[0020] The present invention has the following beneficial effects: (1) The present invention provides a method for restoring bleached corals based on the colonization of zooxanthellae, which is efficient, rapid, and stable, and can fully meet the technical requirements for subsequent colonization of specific highly stress - resistant zooxanthellae and the implementation of coral reef ecological restoration. After coral bleaching, the recovery period is long, and corals are prone to infection and have extremely poor stress resistance, being extremely likely to die in large areas. Based on the co - incubation of zooxanthellae and corals, the present invention can quickly complete the colonization of specific zooxanthellae on bleached corals. This method can directly solve the fundamental problem of the broken symbiotic relationship between hermatypic corals and zooxanthellae in bleached corals, thereby achieving the purpose of quickly restoring bleached corals. At the same time, the inoculation operation of zooxanthellae is relatively simple, with low cost, and has relatively little interference to the coral reef ecosystem.

[0021] (2) The present invention is specifically constructed according to the characteristics of Pocillopora damicornis widely distributed in the South China Sea of our country, and is an important technical support for realizing the breeding of "super corals" based on highly resistant zooxanthellae, which can provide valuable resources for the protection and restoration of the coral reef ecosystem in the South China Sea of our country. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the bleached Pocillopora damicornis; Figure 2The colonization effect diagrams of Symbiodinium after 14 days of colonization in Example 1 and Comparative Example 1, where a is Comparative Example 1 and b is Example 1; Figure 3 The bleaching recovery diagrams of Pocillopora damicornis after 3 months of culture in Example 1 and Comparative Example 1, where a is Comparative Example 1 and b is Example 1. Detailed implementation manners

[0023] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0024] Example 1: A method for restoring bleached Pocillopora damicornis based on inoculation of Symbiodinium includes the following steps: (1) Obtaining of bleached corals Healthy Pocillopora damicornis was domesticated with seawater containing 0.38 mmol / L menthol for 4 days to obtain completely bleached Pocillopora damicornis (microscopic examination showed that algal cells were completely removed). The bleached coral strains were divided into coral branches and domesticated in an indoor culture system with filtered seawater, as Figure 1 shown, and supplemented with Artemia nauplii for feeding daily to maintain their growth activity.

[0025] (2) Preparation of Symbiodinium colonization solution Symbiodinium (Clade C) symbiotic with corals was inoculated into f / 2 medium. After reaching the exponential growth phase, Symbiodinium cells were collected, and then transferred into seawater without f / 2 medium for nutritional starvation treatment for 4 days. The algal cells were collected by low-speed centrifugation (1500 rpm, 5 min), and finally the collected algal cells were poured into a colonization bottle containing seawater, so that the density of Symbiodinium in the colonization bottle was 10 4 cells / mL.

[0026] (3) Co-incubation of bleached corals and Symbiodinium The bleached corals were cultured under a light-dark cycle for 30 days. The culture parameters were: salinity 33 PSU, temperature 25°C, light cycle: dark cycle = 12 h:12 h; under the light cycle culture conditions, the light intensity was 120 μE m -2 s -1 , and the bleached coral branches were regularly transferred into the Symbiodinium colonization solution obtained in step (2). After their tentacles naturally opened, the colonization time was counted for 30 min, and the colonization incubation was carried out 4 times a day. At the same time, Artemia nauplii were supplemented for feeding each time of colonization. After each colonization ended, the coral tissue was immediately transferred back to the coral culture system for continued culture.

[0027] On the 14th day of cyclic cultivation, the symbiotic situation of reef-building corals and zooxanthellae was observed through a microscope. As Figure 2 shown in Figure b in [reference], a large number of symbiotic algae were successfully colonized in the coral polyps.

[0028] (4) After 1 month of co-incubation in step (3), the zooxanthellae-colonized reef-building coral strain has been constructed. At this time, the co-incubation of zooxanthellae and coral is terminated, and the colonized coral is placed in a normal coral culture system to gradually recover. After 3 months, it can be clearly observed that the color and physiological functions of the reef-building coral gradually recover. As Figure 3 shown in Figure b in [reference], the coral has basically returned to normal.

[0029] Example 2: A method for restoring bleached Acropora cervicornis based on inoculation of zooxanthellae, comprising the following steps: (1) Obtaining of bleached corals Healthy Acropora cup-shaped corals were domesticated with seawater containing 0.38 mmol / L menthol for 4 days to obtain completely bleached Acropora cup-shaped corals (microscopic examination showed that algal cells were completely removed). The bleached coral strain was divided into coral branches and domesticated in an indoor culture system with filtered seawater, supplemented with Artemia nauplii for feeding daily to maintain its growth activity.

[0030] (2) Preparation of the zooxanthellae solution for colonization Coral symbiotic zooxanthellae (Clade C) were inoculated into f / 2 medium. After reaching the exponential growth phase, zooxanthellae cells were collected, then transferred into seawater without f / 2 medium for 4 days of nutrient starvation treatment, and the algal cells were collected by low-speed centrifugation (1500 rpm, 5 min). Finally, the collected algal cells were poured into a colonization bottle containing seawater, so that the density of zooxanthellae in the colonization bottle was 5×10 3 cells / mL.

[0031] (3) Co-incubation of bleached corals and zooxanthellae The bleached corals were cultured in a light-dark cycle for 30 days. The culture parameters were: salinity 33 PSU, temperature 25°C, light cycle: dark cycle = 12 h:12 h; under the light cycle culture conditions, the light intensity was 110 μE m -2 s -1 , and the bleached coral branches were regularly transferred into the zooxanthellae solution for colonization obtained in step (2). After their tentacles naturally opened, the colonization was timed for 40 min, incubated 3 times a day, and supplemented with Artemia nauplii for feeding each time of colonization. After each colonization, the coral tissue was immediately transferred back to the coral culture system for continued culture.

[0032] After 1 month of co-incubation in step (3), the symbiotic algal colonization of reef-building coral strains has been completed. At this time, the co-incubation of symbiotic algae and coral is terminated, and the colonized coral is placed in a normal coral culture system to gradually recover.

[0033] Example 3: A method for restoring bleached Acropora cervicornis based on inoculation of symbiotic algae, comprising the following steps: (1) Obtaining bleached corals Healthy Acropora cup corals are domesticated with seawater containing 0.38 mmol / L menthol for 4 days to obtain completely bleached Acropora cup corals (microscopic examination shows that algal cells are 100% removed). The bleached coral strains are divided into coral branches and domesticated in an indoor culture system with filtered seawater, supplemented with Artemia nauplii for feeding daily to maintain their growth activity.

[0034] (2) Preparation of symbiotic algal solution for colonization Symbiotic algae of corals (Clade C) are inoculated into f / 2 medium. After reaching the exponential growth phase, algal cells are collected, then transferred into seawater without f / 2 medium for 4 days of nutrient starvation treatment, and the algal cells are collected by low-speed centrifugation (1500 rpm, 5 min). Finally, the collected algal cells are poured into a colonization bottle containing seawater, so that the density of symbiotic algae in the colonization bottle is 10 4 cells / mL.

[0035] (3) Co-incubation of bleached corals and symbiotic algae The bleached corals are cultured in a light-dark cycle for 30 days. The culture parameters are: salinity 33 PSU, temperature 25 °C, light cycle: dark cycle = 12 h:12 h; under the light cycle culture conditions, the light intensity is 130 μE m -2 s -1 , and the bleached coral branches are regularly transferred into the symbiotic algal solution for colonization obtained in step (2). After their tentacles naturally open, the colonization time is counted for 20 min, and the incubation is carried out 4 times a day. At the same time, Artemia nauplii are supplemented for feeding during each colonization, and the coral tissue is immediately transferred back to the coral culture system for continuous culture after each colonization.

[0036] After 1 month of co-incubation in step (3), the symbiotic algal colonization of reef-building coral strains has been completed. At this time, the co-incubation of symbiotic algae and coral is terminated, and the colonized coral is placed in a normal coral culture system to gradually recover.

[0037] Comparative Example 1: A method for restoring bleached Acropora cervicornis based on inoculation of symbiotic algae, comprising the following steps: (1) Obtaining bleached corals Healthy Pocillopora acuta corals were acclimated with seawater containing 0.38 mmol / L menthol for 6 days to obtain completely bleached Pocillopora acuta corals (microscopic examination showed that 100% of the algal cells were removed). The bleached coral strains were divided into coral branches and acclimated in an indoor culture system with filtered seawater, as Figure 1 shown, and fed with Artemia nauplii daily to maintain their growth activity.

[0038] (2) Preparation of the Symbiodinium inoculum solution Symbiodinium (Clade C) symbiotic with corals was inoculated into f / 2 medium. After it reached the exponential growth phase, Symbiodinium cells were collected, then transferred into seawater without f / 2 medium for 4 days of nutrient starvation treatment. The algal cells were collected by low-speed centrifugation (1500 rpm, 5 min), and finally the collected algal cells were poured into a colonization bottle containing seawater to make the density of Symbiodinium in the colonization bottle 10 2 cells / mL.

[0039] (3) Co-incubation of bleached corals and Symbiodinium The bleached corals were cultured under a light-dark cycle for 30 days. The culture parameters were: salinity 33 PSU, temperature 25 °C, light cycle: dark cycle = 12 h:12 h; under the light cycle culture conditions, the light intensity was 120 μE m -2 s -1 -2, and the bleached coral branches were regularly transferred into the Symbiodinium inoculum solution obtained in step (2). After their tentacles naturally opened, the colonization was timed for 30 min, and the incubation was carried out 4 times a day. At the same time, Artemia nauplii were fed during each colonization. After each colonization, the coral tissue was immediately transferred back to the coral culture system for continued culture.

[0040] On the 14th day of the cycle culture, the symbiotic situation between the reef-building corals and Symbiodinium was observed under a microscope. As Figure 2 shown in Figure a, only an extremely small amount of Symbiodinium successfully colonized in the coral polyps, showing a significant gap compared with Example 1.

[0041] (4) After 1 month of co-incubation in step (3), the Symbiodinium-colonized reef-building coral strain had been constructed. At this time, the co-incubation of Symbiodinium and corals was terminated, and the colonized corals were placed in a normal coral culture system to gradually recover. After 3 months, it could be observed that the color and physiological functions of the reef-building corals were gradually recovering, but the recovery effect was significantly limited. The density of Symbiodinium in the corals was significantly lower than that in Example 1, and the color of the corals was still relatively light, as Figure 3 shown in Figure a.

[0042] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for restoring bleached Acropora based on zooxanthellae inoculation, characterized in that: The following steps are involved: (1) Preparation of solution for colonization with zooxanthellae: First, zooxanthellae are inoculated into a culture medium, and zooxanthellae cells are collected during the exponential growth phase, and then transferred into seawater for nutrient starvation treatment, and finally the zooxanthellae cells are collected by centrifugation and resuspended in seawater to obtain; (2) Co-incubation of bleached corals and zooxanthellae: The bleached corals are cultured in a light-dark cycle, and the bleached corals are regularly transferred to the colonization zooxanthellae solution obtained in step (1) during the light cycle for colonization, thereby completing the recovery of the bleached corals.

2. The method for restoring bleached Acropora based on zooxanthellae inoculation according to claim 1, characterized in that: The culture medium in step (1) is f / 2 culture medium.

3. The method for restoring bleached Acropora based on zooxanthellae inoculation according to claim 1, characterized in that: The duration of the nutritional starvation treatment in step (1) is 3-4 days.

4. The method for restoring bleached Acropora based on zooxanthellae inoculation according to claim 1, characterized in that: The concentration of zooxanthellae in the solution for colonization of zooxanthellae in step (1) is 5×10 3 -5×10 4 cells / mL.

5. The method for restoring bleached Acropora based on zooxanthellae inoculation according to claim 1, characterized in that: The coral is a Staghorn coral.

6. The method for restoring bleached Acropora based on zooxanthellae inoculation according to claim 1, characterized in that: The light-dark cycle culture time in step (2) is 20-40 days, with the light cycle being 10-14 hours per day and the dark cycle being 10-14 hours per day.

7. The method for restoring bleached Acropora based on zooxanthellae inoculation according to claim 1, characterized in that: The colonization conditions in step (2) are as follows: transfer the bleached coral to the colonization zooxanthellae solution during the photoperiod, wait for its tentacles to open naturally, then colonize for 20-40 minutes, and colonize 3-4 times a day.

8. The method for restoring bleached Acropora based on zooxanthellae inoculation according to claim 1, characterized in that: During the planting in step (2), brine shrimps are fed as a supplement.

9. The method for restoring bleached Acropora based on zooxanthellae inoculation according to claim 1, characterized in that: The light intensity of the photoperiod in step (2) is 100-150 μE m -2 s -1 ; The culture parameters for both the light and dark cycles were: salinity 30-35 PSU, temperature 20-30℃.

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