A method for restoring bleached pinnatellacup corals based on inoculation of trichodesmium

Abstract

The application discloses a method for recovering bleached Pocillopora damicornis based on zooxanthella inoculation and belongs to the technical field of coral breeding. The method comprises the following steps: (1) inoculating zooxanthella into a culture medium, collecting zooxanthella cells in the exponential growth phase, then transferring the zooxanthella cells into seawater for nutritional starvation treatment, finally centrifuging the zooxanthella cells and resuspending the zooxanthella cells in seawater to prepare a zooxanthella solution; and (2) performing light-dark cycle cultivation on bleached corals, periodically transferring the bleached corals to the zooxanthella solution for planting in the light period, and completing the recovery of the bleached corals. The method is efficient, rapid and stable, can rapidly complete the planting of specific zooxanthella on bleached corals based on the co-incubation of zooxanthella and corals, solves the fundamental problem of the broken symbiotic relationship between reef-building corals and zooxanthella in bleached corals, realizes the rapid and stable recovery of bleached corals, and plays an important technical support role in the breeding of "super corals" based on high-resistance zooxanthella.

Description

Technical Field

[0001] This invention relates to the field of coral cultivation technology, specifically to a method for restoring albino staghorn corals based on zooxanthellae inoculation. Background Technology

[0002] Coral reef ecosystems are among the most important marine ecosystems in my country, especially in the South China Sea. They are not only biodiversity hotspots but also play an irreplaceable role in maintaining marine ecological balance and fishery resources. However, in recent years, the frequent occurrence of marine heat waves due to global climate change has severely damaged the South China Sea coral reef ecosystem. The increased seawater temperature caused by these heat waves directly triggers large-scale bleaching events in reef-building corals. Coral bleaching is essentially caused by the breakdown of the symbiotic relationship between the host coral and zooxanthellae. Zooxanthellae, as single-celled algae, live in the endoderm cells of corals and provide energy and nutrients to the host corals through photosynthesis. However, when seawater temperatures rise abnormally, the photosynthetic system of zooxanthellae is disrupted, producing excessive reactive oxygen species, which damage the host coral cells. To protect themselves, the host corals expel zooxanthellae, thus losing their primary energy source, ultimately leading to coral bleaching and even death.

[0003] Large-scale coral bleaching not only causes corals to lose their color but also severely weakens their survival ability and ecological function. Bleached reef-building corals are more susceptible to disease, experience slower growth, reduced reproductive capacity, and may even lead to the collapse of the entire coral reef ecosystem. Coral reef degradation in the South my country Sea has already had a significant impact on marine biodiversity and fishery resources, while also threatening the livelihoods of coastal communities and the safety of the coastline. Faced with this serious situation, strengthening the protection and management of coral reefs is imperative. Therefore, in addition to long-term measures such as reducing greenhouse gas emissions and controlling marine pollution, developing a rapid and effective coral bleaching restoration technology is particularly urgent.

[0004] Currently, common international methods for coral bleaching restoration include coral transplantation and artificial reef construction. Coral transplantation involves transplanting healthy coral fragments to degraded areas to promote coral community recovery. Artificial reef construction involves placing artificial structures to provide an attachment substrate for coral larvae, helping the coral population rebuild. However, these methods all have certain limitations. While coral transplantation and artificial reef construction can improve coral coverage in localized areas in the short term, they are costly, and transplanted corals may not adapt to the new environmental conditions, resulting in limited restoration effects. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a method for restoring bleached staghorn corals based on zooxanthellae inoculation, thereby solving the problems of high cost, potential inability of transplanted corals to adapt to new environmental conditions, and limited restoration effects of existing coral bleaching methods.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method for restoring albino staghorn corals based on zooxanthellae inoculation includes the following steps: (1) Preparation of the solution of colonizing phytoxanthin: First, zooxanthellae are inoculated into a culture medium. During the exponential growth phase, zooxanthellae cells are collected. Then, they are transferred to seawater for nutrient starvation treatment. Finally, the zooxanthellae cells are collected by centrifugation and resuspended in seawater to obtain the product. (2) Co-incubation of albino corals and zooxanthellae: The albino corals were cultured in a light-dark cycle. During the light cycle, the albino corals were periodically transferred to the colonization zooxanthellae solution obtained in step (1) for colonization, thus completing the recovery of the albino corals.

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

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

[0009] Furthermore, the nutritional starvation treatment in step (1) lasts for 3-4 days.

[0010] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: This invention, by subjecting zooxanthellae cells to nutritional starvation treatment, causes them to consume excess stored nutrients, thereby stimulating a better transformation from a free state to a symbiotic state.

[0011] Furthermore, the concentration of zooxanthellae in the zooxanthellae solution in step (1) is 5 × 10⁻⁶. 3 -5×10 4 Cells / mL.

[0012] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: By optimizing the cell concentration in the zooxanthellae concentrate, the present invention promotes the colonization efficiency of zooxanthellae and achieves a highly efficient and ideal colonization effect.

[0013] Furthermore, the coral is a staghorn cup-shaped coral.

[0014] Furthermore, in step (2), the light-dark cycle culture time is 20-40 days, the light cycle is 10-14 hours per day, and the dark cycle is 10-14 hours per day.

[0015] Preferably, the light-dark cycle culture time in step (2) is 30 days, with a light cycle of 12 hours per day and a dark cycle of 12 hours per day.

[0016] Furthermore, the conditions for colonization in step (2) are as follows: transfer the albino coral to the colonization zooxanthellae solution during the photoperiod, wait for its tentacles to open naturally, and then colonize for 20-40 minutes, 3-4 times a day.

[0017] Furthermore, brine shrimp are fed during the colonization process in step (2).

[0018] Furthermore, in step (2), the photoperiodic light intensity is 100-150 μE m. -2 s -1 The cultivation parameters for both photoperiod and darkperiod were: salinity 30-35 PSU, temperature 20-30℃.

[0019] Preferably, the photoperiodic light intensity in step (2) is 120 μE m. -2 s -1 The culture parameters for both the light and dark cycles were: salinity 33 PSU, temperature 25℃.

[0020] The present invention has the following beneficial effects: (1) This invention provides a method for restoring bleached corals based on zooxanthellae colonization. This method is efficient, rapid, and stable, and can fully meet the technical requirements for subsequent colonization of specific highly resilient zooxanthellae and the development of coral reef ecological restoration. After coral bleaching, the recovery period is long, and corals are susceptible to disease, exhibiting extremely poor resilience and prone to large-scale mortality. This invention, based on the co-incubation of zooxanthellae and corals, can rapidly complete the colonization of bleached corals with specific zooxanthellae. This method can directly solve the fundamental problem of the broken symbiotic relationship between reef-building corals and zooxanthellae in bleached corals, thereby achieving the goal of rapid restoration of bleached corals. Simultaneously, the zooxanthellae inoculation operation is relatively simple, low-cost, and causes minimal disturbance to the coral reef ecosystem.

[0021] (2) This invention is specifically designed for the characteristics of the staghorn cup coral that is widely distributed in the South my country Sea. It is an important technical support for the breeding of "super corals" based on highly resistant zooxanthellae and can provide valuable resources for the protection and restoration of coral reef ecosystems in the South my country Sea. Attached Figure Description

[0022] Figure 1 It is an albino staghorn cup-shaped coral; Figure 2The images show the colonization effect of Zooxanthus algae after 14 days in Example 1 and Comparative Example 1, where a is Comparative Example 1 and b is Example 1. Figure 3 The images show the bleaching recovery of staghorn cup corals after 3 months of cultivation in Example 1 and Comparative Example 1, where a is Comparative Example 1 and b is Example 1. Detailed Implementation

[0023] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0024] Example 1: A method for restoring albino staghorn corals based on zooxanthellae inoculation includes the following steps: (1) Acquisition of albino corals Healthy staghorn cup corals were acclimated for 4 days in seawater containing 0.38 mmol / L menthol to obtain completely bleached staghorn cup corals (microscopic examination showed 100% removal of algal cells). The bleached coral strains were then separated into coral fragments and acclimated in an indoor culture system using filtered seawater. Figure 1 As shown, they are supplemented with brine shrimp daily to maintain their growth and activity.

[0025] (2) Preparation of the solution of colonizing phytoxanthin Coral symbiotic zooxanthellae (Clade C) were inoculated into f / 2 medium. After reaching the exponential growth phase, zooxanthellae cells were collected and then transferred to seawater without f / 2 medium for nutrient starvation treatment for 4 days. Algal cells were collected by low-speed centrifugation (1500 rpm, 5 min). Finally, the collected algal cells were poured into colonization bottles containing seawater, ensuring a zooxanthellae density of 10-1. 4 Cells / mL.

[0026] (3) Co-incubation of albino corals and zooxanthellae Albino corals were cultured under photo-dark cycle conditions for 30 days with the following parameters: salinity 33 PSU, temperature 25℃, and photo-dark cycle = 12 h: 12 h. Under photo-dark cycle conditions, the light intensity was 120 μE m. -2 s -1 The bleached coral fragments were periodically transferred into the colonized zooxanthellae solution obtained in step (2). After the tentacles opened naturally, the colonization time was 30 minutes. The colonization and incubation were carried out 4 times a day. At the same time, brine shrimp were fed during each colonization. After each colonization, the coral tissue was immediately transferred back to the coral culture system for continued culture.

[0027] On day 14 of cyclic culture, the symbiotic relationship between reef-building corals and zooxanthellae was observed under a microscope, such as... Figure 2 As shown in Figure b, a large number of symbiotic algae have successfully colonized the coral polyp.

[0028] (4) After a one-month co-incubation period in step (3), the zooxanthellae colonization of the reef-building coral system has been completed. At this point, the co-incubation of the zooxanthellae and coral is terminated, and the colonized coral is placed in a normal coral culture system to allow it to gradually recover. After three months, the color and physiological functions of the reef-building coral can be clearly observed to gradually recover, such as... Figure 3 As shown in Figure b, the coral has basically recovered to normal.

[0029] Example 2: A method for restoring albino staghorn corals based on zooxanthellae inoculation includes the following steps: (1) Acquisition of albino corals Healthy staghorn cup corals were acclimated for 4 days in seawater containing 0.38 mmol / L menthol to obtain completely bleached staghorn cup corals (microscopic examination showed that 100% of algal cells were removed). The bleached coral strains were separated into coral fragments and placed in an indoor culture system using filtered seawater for acclimation. They were supplemented with brine shrimp daily to maintain their growth activity.

[0030] (2) Preparation of the solution of colonizing phytoxanthin Coral symbiotic zooxanthellae (Clade C) were inoculated into f / 2 medium. After reaching the exponential growth phase, zooxanthellae cells were collected and then transferred to seawater without f / 2 medium for nutrient starvation treatment for 4 days. Algal cells were collected by low-speed centrifugation (1500 rpm, 5 min). Finally, the collected algal cells were poured into colonization bottles containing seawater, ensuring a zooxanthellae density of 5 × 10⁻⁶ cells / ml. 3 Cells / mL.

[0031] (3) Co-incubation of albino corals and zooxanthellae Albino corals were cultured under photo-dark cycle conditions for 30 days with the following parameters: salinity 33 PSU, temperature 25℃, and photo-dark cycle = 12 h: 12 h. Under photo-dark cycle conditions, the light intensity was 110 μE m. -2 s -1 The bleached coral fragments were periodically transferred into the colonized zooxanthellae solution obtained in step (2). After the tentacles opened naturally, the colonization time was 40 minutes. The colonization was carried out 3 times a day. At the same time, brine shrimp were fed during each colonization. After each colonization, the coral tissue was immediately transferred back to the coral culture system for continued culture.

[0032] (4) After a month of co-incubation in step (3), the zooxanthellae colonization of the reef-building coral system has been completed. 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 allow it to gradually recover.

[0033] Example 3: A method for restoring albino staghorn corals based on zooxanthellae inoculation includes the following steps: (1) Acquisition of albino corals Healthy staghorn cup corals were acclimated for 4 days in seawater containing 0.38 mmol / L menthol to obtain completely bleached staghorn cup corals (microscopic examination showed that 100% of algal cells were removed). The bleached coral strains were separated into coral fragments and placed in an indoor culture system using filtered seawater for acclimation. They were supplemented with brine shrimp daily to maintain their growth activity.

[0034] (2) Preparation of the solution of colonizing phytoxanthin Coral symbiotic zooxanthellae (Clade C) were inoculated into f / 2 medium. After reaching the exponential growth phase, zooxanthellae cells were collected and then transferred to seawater without f / 2 medium for nutrient starvation treatment for 4 days. Algal cells were collected by low-speed centrifugation (1500 rpm, 5 min). Finally, the collected algal cells were poured into colonization bottles containing seawater, ensuring a zooxanthellae density of 10-1. 4 Cells / mL.

[0035] (3) Co-incubation of albino corals and zooxanthellae Albino corals were cultured under photo-dark cycle conditions for 30 days with the following parameters: salinity 33 PSU, temperature 25℃, and photo-dark cycle = 12 h: 12 h. Under photo-cycle conditions, the light intensity was 130 μE m. -2 s -1 The bleached coral fragments were periodically transferred into the colonized zooxanthellae solution obtained in step (2). After the tentacles opened naturally, the colonization time was 20 minutes. The colonization was carried out 4 times a day. At the same time, brine shrimp were fed during each colonization. After each colonization, the coral tissue was immediately transferred back to the coral culture system for continued culture.

[0036] (4) After a month of co-incubation in step (3), the zooxanthellae colonization of the reef-building coral system has been completed. 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 allow it to gradually recover.

[0037] Comparative Example 1: A method for restoring albino staghorn corals based on zooxanthellae inoculation includes the following steps: (1) Acquisition of albino corals Healthy staghorn cup corals were acclimated for 6 days in seawater containing 0.38 mmol / L menthol to obtain completely bleached staghorn cup corals (microscopic examination showed 100% removal of algal cells). The bleached coral strains were then separated into coral fragments and acclimated in an indoor culture system using filtered seawater. Figure 1 As shown, they are supplemented with brine shrimp daily to maintain their growth and activity.

[0038] (2) Preparation of the solution of colonizing phytoxanthin Coral symbiotic zooxanthellae (Clade C) were inoculated into f / 2 medium. After reaching the exponential growth phase, zooxanthellae cells were collected and then transferred to seawater without f / 2 medium for nutrient starvation treatment for 4 days. Algal cells were collected by low-speed centrifugation (1500 rpm, 5 min). Finally, the collected algal cells were poured into colonization bottles containing seawater, ensuring a zooxanthellae density of 10-1. 2 Cells / mL.

[0039] (3) Co-incubation of albino corals and zooxanthellae Albino corals were cultured under photo-dark cycle conditions for 30 days with the following parameters: salinity 33 PSU, temperature 25℃, and photo-dark cycle = 12 h: 12 h. Under photo-dark cycle conditions, the light intensity was 120 μE m. -2 s -1 The bleached coral fragments were periodically transferred into the colonized zooxanthellae solution obtained in step (2). After the tentacles opened naturally, the colonization time was 30 minutes. The colonization was carried out 4 times a day. At the same time, brine shrimp 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 day 14 of cyclic culture, the symbiotic relationship between reef-building corals and zooxanthellae was observed under a microscope, such as... Figure 2 As shown in Figure a, only a very small amount of zooxanthellae successfully colonized the coral polyps, which is significantly different from Example 1.

[0041] (4) After a one-month co-incubation period in step (3), the zooxanthellae colonized reef-building coral strain has been established. At this point, the co-incubation of zooxanthellae and coral is terminated, and the colonized coral is placed in a normal coral culture system to allow it to gradually recover. After three months, the color and physiological functions of the reef-building coral can be observed to gradually recover, but the recovery effect is obviously limited. The density of zooxanthellae in the coral is significantly lower than in Example 1, and the color of the coral is still relatively light. Figure 3 As shown in Figure a.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for restoring albino staghorn cup corals based on zooxanthellae inoculation, characterized in that, Includes the following steps: (1) Preparation of the solution of colonizing phytoxanthin: First, zooxanthellae were inoculated into a culture medium. During the exponential growth phase, zooxanthellae cells were collected and then transferred to seawater for nutrient starvation treatment for 3-4 days. Finally, the zooxanthellae cells were collected by centrifugation and resuspended in seawater to obtain the culture medium. The concentration of zooxanthellae in the colonized zooxanthellae solution was 5 × 10⁻⁶. 3 -5×10 4 cells / mL; (2) Co-incubation of albino corals and zooxanthellae: The albino corals were cultured in a light-dark cycle. During the light cycle, the albino corals were periodically transferred to the zooxanthellae solution obtained in step (1) for colonization to complete the recovery of the albino corals. The colonization conditions were as follows: during the light cycle, the albino corals were transferred to the zooxanthellae solution, and after their tentacles opened naturally, the colonization time was 20-40 minutes. The colonization was carried out 3-4 times a day. The coral in question is a staghorn cup-shaped coral.

2. The method for restoring albino staghorn cup corals based on zooxanthellae inoculation according to claim 1, characterized in that, The culture medium used in step (1) is f / 2 culture medium.

3. The method for restoring albino staghorn cup corals based on zooxanthellae inoculation according to claim 1, characterized in that, In step (2), the light-dark cycle culture time is 20-40 days, with a light cycle of 10-14 h per day and a dark cycle of 10-14 h per day.

4. The method for restoring albino staghorn cup corals based on zooxanthellae inoculation according to claim 1, characterized in that, In step (2), brine shrimp are used as feed during the colonization process.

5. The method for restoring albino staghorn cup corals based on zooxanthellae inoculation according to claim 1, characterized in that, In step (2), the photoperiodic light intensity is 100-150 μE m. -2 s -1 The cultivation parameters for both photoperiod and darkperiod were: salinity 30-35 PSU, temperature 20-30℃.

Images