Method for enhancing adhesion ability of coral larvae after enhancement and release

Abstract

The invention discloses a method for enhancing adhesion ability of coral larvae after enhancement and release. According to the method, coral larvae (such as coral plankton larvae) are exposed to a 40 mM calcium chloride (CaCl2) solution to be treated for 12-24 hours, and then enhancement and release are carried out. The treated larvae show remarkably enhanced adhesion ability after releasing, the accumulated adhesion rate within 3 days reaches 66.33% and is increased by 240% compared with that of a control group in the same period, and the average survival rate reaches 98.14%. Meanwhile, the transverse growth area and the budding number of attached larvae are remarkably increased, and the density of zooxanthellae, the photosynthetic efficiency and the chlorophyll content have no negative effects. The CaCl2 solution is used for successfully inducing the continuous attachment effect of the coral larvae, the method is easy and convenient to operate, low in cost and environmentally friendly, the attachment success rate of enhancement and release of the coral larvae and the subsequent growth performance of the larvae are remarkably increased, and the method is suitable for repairing and protecting a coral reef ecological system.

Description

Technical Field

[0001] This invention relates to the field of coral reef ecological restoration technology, specifically to a method for enhancing the attachment ability of coral larvae after restocking and release. Background Technology

[0002] Coral reefs are among the most productive and biodiverse ecosystems in the ocean, but they are facing severe global degradation. Against this backdrop, artificial restoration and resource recovery of coral reefs are crucial. Among these methods, the release of coral planktonic larvae is a key way to directly replenish coral populations; its success depends heavily on the larvae's attachment success rate on selected natural substrates and their survival and growth after attachment.

[0003] Currently, some methods exist for promoting larval attachment to artificial substrates in controlled nursery stages. However, these traditional methods have significant limitations and inapplicability when applied to stock enhancement and release practices. For example, bio-inducers such as crustose coralline algae (CCA) or specific bacterial strains (such as Metabacillus sp. cB07 mentioned in this invention) can effectively induce attachment, but their cultivation conditions are demanding, the cycle is long, the cost is high, and the effect is greatly affected by environmental factors and the organism's own condition, resulting in low stability and standardization. More importantly, in the open marine environment of stock enhancement, it is difficult to continuously provide and maintain the effective concentration of such bio-inducers, causing their attachment-promoting effect to not continue after release. Although some research exists on non-biochemical inducers, existing technologies mostly rely on continuous exposure of larvae throughout the entire attachment period. This model is not only complex and costly to operate, but may also have negative effects such as toxic inhibition on subsequent larval development due to long-term contact. More importantly, this "continuous treatment" model is neither economical nor practical in open marine environments.

[0004] In summary, a significant gap exists in the current technological field: the lack of an effective method specifically designed to enhance the attachment and survival capabilities of coral larvae after restocking. Most existing methods focus on attachment in laboratory settings, failing to address the core challenge of maintaining high attachment potential and healthy growth in the absence of continuous induction after larvae leave the controlled environment and enter natural marine environments. Therefore, there is an urgent need in this field for a simple and sustained-effect attachment-promoting technology. This method should enable coral larvae to autonomously and efficiently attach themselves after release into natural marine environments, even without external inducers, through short-term pretreatment, ensuring their subsequent survival and growth, thereby truly improving the success rate of restocking. Summary of the Invention

[0005] This invention aims to solve the problems of low attachment rate, unstable survival rate and slow growth of coral larvae after release and propagation in coral reef restoration, and provides a method for promoting the attachment of coral larvae based on the "continuous attachment effect".

[0006] The first objective of this invention is to provide a method for enhancing the attachment ability of coral larvae after propagation and release, comprising the following steps: placing coral planktonic larvae in an attachment promoter solution for short-term exposure treatment, then transferring them to filtered seawater without the attachment promoter for cultivation and / or releasing them into natural sea areas to induce them to complete attachment under conditions where the attachment promoter is not continuously present, wherein the attachment promoter is a 30-50 mM calcium chloride solution.

[0007] Preferably, the concentration of the calcium chloride solution is 40 mM.

[0008] Preferably, the calcium chloride solution is prepared using filtered seawater.

[0009] Preferably, the filtered seawater is 0.2-micron filtered seawater.

[0010] Preferably, the duration of the short-term exposure treatment is 6 to 72 hours.

[0011] Preferably, the duration of the short-term exposure treatment is 12 to 24 hours.

[0012] Preferably, the coral is a staghorn cup coral (Pocillopora damicornis).

[0013] Preferably, the culture conditions for the coral larvae are: temperature 26.0±0.2℃, light-dark cycle 12 h:12 h, and light intensity 200 μmol·photons·m. -2 ·s -1 Density ≤ 1 piece / ml.

[0014] Preferably, the specific steps are as follows:

[0015] A. Larval Collection and Temporary Rearing: Healthy floating larvae of Pocilloporadamicornis coral (Pocilloporadamicornis) are collected during the peak period of coral larval emergence and temporarily reared.

[0016] B. Preparation of adhesion promoter: Prepare a 30-50 mM calcium chloride solution as the adhesion promoter working solution;

[0017] C. Short-term exposure treatment: Place the planktonic larvae obtained in step A into the working solution of the adhesion promoter in step B for short-term exposure treatment for 12-24 hours;

[0018] D. Continuing culture and evaluation: Transfer the larvae treated in step C to conventional filtered seawater or natural seawater without attachment promoters and continue to culture for 1-3 days;

[0019] E. Release and propagation: Release the treated larvae.

[0020] Preferably, the calcium chloride solution is a 40 mM calcium chloride solution prepared by filtering seawater through a 0.2-micron filter, and the temporary rearing is carried out under laboratory conditions. The environmental conditions for culturing the planktonic larvae of the staghorn cup coral are: temperature 26.0 ± 0.2℃, light-dark cycle 12 h:12 h, and light intensity 200 μmol·photons·m -2 ·s -1 Density ≤ 1 piece / ml.

[0021] The short-term exposure treatment can induce coral larvae to produce a continuous attachment effect.

[0022] In step D, the attachment rate monitored during the 3-day extended culture period was significantly higher than that of the control group without the attachment promoter. After the larvae attached and continued to be cultured, the lateral growth area and / or number of buds of the attached larvae were significantly higher than those of the control group on the 14th day of culture.

[0023] After the larvae underwent short-term exposure treatment were transferred to the propagation and release site, the attachment rate reached 66.33% on the third day.

[0024] The coral described in this invention is the staghorn coral (Pocillopora damicornis), but its application is not limited to other reef-building coral species.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. Pioneering "Continuous Attachment Effect": This invention is the first to discover and apply the "continuous induction" effect of low-concentration calcium chloride on coral larvae attachment. Only a short exposure of 12-24 hours is needed to produce a sustained effect on larval attachment behavior, enabling them to maintain a very high attachment desire even after being moved into normal seawater. This breaks with the traditional understanding that larvae must be continuously exposed to the inducing environment, representing a fundamental technological breakthrough.

[0027] 2. Significantly improved attachment and survival rates: Larvae transferred after treatment with 40 mM calcium chloride for 12-24 hours showed an attachment rate of 18.89% to 51.11% within 3 days, significantly higher than all control groups including shell-shaped coral algae (CCA), probiotics (Metabacillus sp. cB07), negative control (sodium chloride), and blank control (filtered seawater) (p < 0.05), and the average larval survival rate was as high as 98.14%.

[0028] 3. Promotes subsequent growth and development: Larvae treated with the method of this invention exhibit superior growth advantages after attachment. By day 14, their lateral growth area (3.50 cm²) 2 The number of individuals and budding numbers (2.34 hydras / individual) were significantly higher than those in the control group, indicating that the induction effect not only promotes attachment but also has a long-term positive impact on early development.

[0029] 4. In the simulated propagation and release scenario, the cumulative attachment rate of larvae in the CaCl2-treated group reached 66.33% within 3 days, which was significantly higher than that in the control group, proving that this method has the potential for application in real marine environments.

[0030] 5. Simple operation, low cost, and easy to promote: Compared with CCA cultivation or probiotic induction, this method only requires short-term chemical treatment, without complex equipment or continuous intervention, and is suitable for large-scale coral reef restoration projects.

[0031] 6. This method provides a new technological paradigm for stock enhancement and release: It allows the separation of the "attachment treatment" from the "transportation / release" process. Larvae can undergo short-term exposure treatment in a shore-based facility, and then be transported to the release point or attach in their natural environment without the need to maintain a complex induction environment, greatly increasing the flexibility and success rate of stock enhancement and release.

[0032] This invention provides a method for propagation and release of staghorn cup coral (Pocilloporadamicornis) larvae by inducing a sustained attachment effect through short-term exposure to an attachment promoter, significantly improving larval attachment rate, survival rate, and growth performance. By exposing coral larvae to a specific attachment promoter (especially calcium chloride solution) for a short period, a sustained attachment effect can be stimulated. Even after the promoter is subsequently removed, the attachment rate of larvae and the growth performance of attached larvae in the subsequent non-inducing environment are still significantly improved, thus providing a novel, efficient, and reliable solution to the technical problem of low attachment efficiency in coral propagation and release. Attached Figure Description

[0033] Figure 1 This is a flowchart of the experimental design for evaluating the adhesion persistence effect of the present invention.

[0034] Figure 2 This is a graph showing the results of the adhesion persistence effect evaluation test of the present invention. A represents 0.2-micron filtered seawater (control group), 80 mM NaCl, shell-like coral algae (CCA), and 1×10⁻⁶... 7The results of the evaluation of the persistence effect of treatment with Metabacillus sp. cB07 bacterial solution and 40mM CaCl2 solution on coral larvae (including attachment rate and survival rate); B represents the persistence of coral larvae attachment after a short exposure of 12 hours and subsequent transfer to filtered seawater for 1 day of culture (arrows indicate attached coral larvae).

[0035] Figure 3 This is a diagram showing the results of the experimental assessment of the growth and physiology of the attached larvae of the staghorn cup-shaped coral according to the present invention.

[0036] Figure 4 This is a diagram showing the attachment results of the propagation and release experiment of the treated staghorn cup coral larvae according to the present invention. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0038] This invention uses staghorn cup coral larvae for experiments, with culture conditions of 26.0±0.2℃, a light-dark cycle of 12h:12h, and a light intensity of 200 μmol·photons·m. -2 ·s -1 Cultivate larvae at a density of ≤1 larva / ml.

[0039] The 0.2-micron filtered seawater used in this invention refers to seawater filtered by a filter material with a pore size of 0.2 microns.

[0040] Example 1: Assessment of larval continuous attachment effect and larval growth

[0041] (1) Material preparation

[0042] Healthy adult staghorn corals (Pocillopora damicornis) were collected from the Luhuitou waters of Sanya City, Hainan Province, China, between May and August 2023 and 2024. Planktonic larvae were collected during the peak larval release period (from the fifth to the tenth day of the first lunar month). The larvae were temporarily cultured in a laboratory using 0.2-micron filtered seawater, maintained at a temperature of 26.0 ± 0.2℃, with a 12 h:12 h light-dark cycle and a light intensity of 200 μmol·photons·m. -2 ·s -1 The density was ≤1 organism / ml. The experiment was conducted at the Hainan Tropical Marine Biology Experimental Station of the South China Sea Institute of Oceanology, Chinese Academy of Sciences.

[0043] Concurrently, *Hydrolithon reinboldii* was collected and cut into 0.5 × 0.5 cm pieces for later use. Working solutions of 80 mM NaCl and 40 mM CaCl2 were prepared using filtered seawater. After culturing strain *Metabacillus sp. cB07*, the seawater was resuspended using a filter medium with a pore size of 0.2 micrometers to prepare a solution with a concentration of 1 × 10⁻⁶. 7 Bacterial solution with cells / mL.

[0044] (2) Assessment of the continuous adhesion effect

[0045] like Figure 1 As shown, this invention sets up five experimental working solutions: filtered seawater (control), 80 mM NaCl, 0.5 × 0.5 cm shell-like coral algae (CCA), and 1 × 10 7 Metabacillus sp. cB07 bacterial culture (cells / mL) and 40 mM CaCl2 solution were used. Healthy staghorn cup coral planktonic larvae (≤1000 larvae) were exposed to the working solution (1000 mL) of each treatment group. At 6, 12, 24, 48, and 72 hours, 50 unattached larvae were transferred to 50 mL of 0.2 micron filtered seawater for further culture (each group had 3 replicates; culture conditions were: 26.0±0.2℃, 12 h:12 h light / dark cycle, light intensity 200 μmol·photons·m -2 ·s -1 The adhesion rate and survival rate were recorded on days 1, 2, and 3 after transfer at different time points to evaluate the persistence effect of the four adhesion promoters.

[0046] The results are as follows Figure 2 As shown, the larvae treated with 40 mM CaCl2 for 12-24 hours had an attachment rate of 18.89%-51.11% within 3 days after being transferred to filtered seawater, which was significantly higher than that of other groups (0-18.89%) (p < 0.05), and the survival rate was also high (average 98.14%).

[0047] (3) Assessment of the growth and photophysiological status of the attached larvae

[0048] In step (2), the five treatment groups, after 12 hours of exposure and on day 1 after transfer, were continuously cultured for 14 days using 0.2-micron filtered seawater (each group had 3 replicates; the culture conditions were: 26.0±0.2℃, 12 h:12 h light-dark cycle, and 200 μmol·photons·m² light intensity). -2 ·s -1Survival rate, number of buds, growth area, and maximum photosynthetic efficiency of larvae were monitored on days 1, 7, and 14 of culture; zooxanthellae density, chlorophyll content, and calcification rate of larvae were monitored on day 14 of culture.

[0049] The results are as follows Figure 3 As shown: the survival rate of larvae in the CaCl2-treated group was not significantly different from that in the control group; by day 14, their lateral growth area (3.50 cm²) was... 2 The number of individuals and buds (2.34 polyps / individual) were significantly higher than those of the control group (p < 0.05), and there were no significant negative effects on zooxanthellae density, maximum photosynthetic efficiency and chlorophyll content.

[0050] Example 2: Assessment of larval attachment ability after propagation and release

[0051] This embodiment aims to simulate a stock enhancement and release scenario to evaluate the attachment ability of larvae exposed to a short-term attachment promoter under such conditions. Following the method in Example 1, healthy planktonic larvae were exposed to five treatment groups for 12 hours. The larvae from the treatment and control groups were then introduced into aquariums (30 × 18 × 20 cm) containing flowing sand-filtered seawater (simulating natural ocean currents) (each group had 3 replicates; culture conditions were: 26.0 ± 0.2℃, 12 h:12 h light / dark cycle, and 200 μmol·photons·m² light intensity). -2 ·s -1 Within 3 days after the larvae were released, the attachment rate of larvae was recorded and calculated daily.

[0052] The results are as follows Figure 4 As shown, the larvae in the 40 mM CaCl2-treated group maintained a significantly high attachment rate of 48.67% on the first day of restocking, which was significantly higher than that of other treatment groups and the control group during the same period (p < 0.05). Furthermore, the cumulative attachment rate of the CaCl2-treated group larvae within 3 days was 66.33%, 240% higher than that of the control group during the same period (p < 0.05). This simulated restocking experiment confirms that staghorn cup coral larvae treated with 40 mM CaCl2 for a short period exhibit significantly enhanced attachment ability and settlement success rate under conditions closer to the real marine environment, verifying the effectiveness and application potential of the method of this invention in coral reef restocking practices.

[0053] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for enhancing the attachment ability of coral larvae after restocking, characterized in that, The method comprises the following steps: After short-term exposure treatment of the coral planulae in the solution of the adhesion-promoting agent, the planulae are transferred to filtered seawater without the adhesion-promoting agent for culture and / or released in natural sea areas to induce the planulae to complete adhesion without the continuous presence of the adhesion-promoting agent, which is a 30-50 mM calcium chloride solution.

2. The method of claim 1, wherein, The concentration of the calcium chloride solution is 40 mM.

3. The method according to claim 1 or 2, characterized in that, The calcium chloride solution is prepared using filtered seawater.

4. The method of claim 3, wherein, The filtered seawater is 0.2-micron filtered seawater.

5. The method of claim 1, wherein, The short-term exposure treatment lasts for 6-72 hours.

6. The method of claim 5, wherein, The short-term exposure treatment lasts for 12-24 hours.

7. The method of claim 1, wherein, The coral is Pocillopora damicornis.

8. The method of claim 1, wherein, The culture condition of the coral larvae is temperature 26.0±0.2℃, light and dark cycle 12 h:12 h, light intensity 200 μmol·photons·m -2 ·s -1 , density ≤1 per milliliter.

9. The method of claim 1, wherein, The specific steps are as follows: A. Larvae collection and temporary culture: collect healthy Pocillopora damicornis planulae during the peak of coral larval release and temporarily culture the planulae; B. Preparation of adhesion-promoting agent: prepare a 30-50 mM calcium chloride solution as the adhesion-promoting agent working solution; C. Short-term exposure treatment: place the planulae obtained in step A in the adhesion-promoting agent working solution of step B for short-term exposure treatment for 12-24 hours; D. Continued culture and evaluation: transfer the planulae treated in step C to regular filtered seawater or natural seawater without the adhesion-promoting agent for continued culture for 1-3 days; E. Release for propagation: release the treated planulae.

10. The method of claim 9, wherein, The calcium chloride solution was a 40 mM calcium chloride solution prepared from 0.2 micron filtered seawater, the temporary rearing was temporary rearing under laboratory conditions, and the environmental conditions for culturing the Pocillopora damicornis planulae were: temperature 26.0 ± 0.2 °C, light-dark cycle 12 h: 12 h, light intensity 200 μmol·photons·m -2 ·s -1 , density ≤ 1 per ml.

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