Bacterium of the family flavobacteriaceae with high efficiency promoting effect on metamorphosis of coral larvae attachment and application thereof

Abstract

This invention discloses a Flavobacteriaceae bacterium that highly promotes the attachment and metamorphosis of coral larvae and its applications. Flavobacteriaceae SCSIO 2518 was deposited on November 14, 2025, at the Guangdong Provincial Microbial Culture Collection Center, No. 100, Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC No. 67297. This invention utilizes Flavobacteriaceae 2518, isolated and purified from calcified algae collected from the Luhuitou coral reef area in Sanya, Hainan Province, China. This bacterium significantly improves the attachment rate and metamorphosis rate of stony coral larvae, shortens the time required, and has no toxic side effects. Therefore, this invention can be applied to promote the attachment and metamorphosis of coral larvae, facilitate large-scale artificial coral breeding, provide high-quality seedling resources for coral population recovery, and thus promote the restoration of degraded coral reef ecosystems.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Flavobacterium bacteria that promotes the attachment and metamorphosis of coral larvae and its applications. Background Technology

[0002] Coral reef ecosystems are among the world's most biodiverse ecosystems, providing survival (including habitat, foraging, and reproduction) for approximately 25% of marine species and offering coastal protection, food, and economic (fisheries, tourism, etc.) resources for about one billion people globally. Furthermore, the rich biological resources within coral reef ecosystems offer significant potential for the development of novel pharmaceuticals. However, in recent decades, global coral reef ecosystems have suffered severe degradation due to climate change and human activities (overfishing, pollution, etc.). In particular, marine heat waves caused by global warming have resulted in several large-scale bleaching events of stony corals. As of 2025, 84% of the world's coral reef ecosystems have been affected by bleaching events, covering at least 82 countries and regions. Research is urgently needed to promote the recovery of stony coral populations, thereby accelerating the restoration of coral reef ecosystems.

[0003] Larval attachment and metamorphosis are core bottlenecks in the life cycle of stony corals, and their success rate directly affects the efficiency of larval replenishment and spatial distribution patterns, serving as a prerequisite for the effective recovery of stony coral populations. However, this crucial process is highly dependent on exogenous induction signals: in the natural environment, the lack of specific induction signals not only significantly prolongs the planktonic period but may even lead to complete cessation of attachment, ultimately resulting in larval replenishment failure and becoming a core obstacle in the current coral reef ecosystem restoration process. Therefore, developing methods to efficiently improve the attachment and metamorphosis rate of coral larvae is crucial for accelerating coral population recovery and coral reef ecosystem restoration.

[0004] Previous studies have confirmed that *Pseudomonas* spp. Pseudoalteromonas These bacteria possess inducing abilities; their biofilms or metabolites, such as tetrabromopyrrole, can efficiently promote coral larval metamorphosis. However, their effect on increasing attachment rates is limited, restricting their application in coral reef restoration practices. In recent years, with the development of high-throughput sequencing technology, correlation analysis between bacterial abundance and coral larval attachment and metamorphosis rates has revealed a large number of potential coral larval attachment and metamorphosis-inducing bacterial resources in the natural environment. However, current research on these potential microbial resources has significant shortcomings—only focusing on the genus *Tetranychus* (…). Thalassomonas ), bright rose-colored fungi ( Roseivivax ), genus *Pseudomonas* Pseudovibrio Acinetobacter spp. Acinetobacter ) and Micrococcus genus ( Microbulbifer ), Bacillus spp. Bacillus ), Phytobacter genus,Salipiger A few strains of this genus have been shown to increase the attachment and metamorphosis rate of coral larvae, but the functions of the vast majority of potential inducing bacteria have not yet been verified, let alone transformed into practically applicable technical resources. Therefore, screening for more strains with highly efficient inducing effects on coral larvae attachment and metamorphosis will not only enrich the resource pool of coral larvae inducing bacteria, but also provide key technical support for overcoming the bottleneck of larval replenishment, promoting coral population recovery, and coral reef ecosystem restoration, which has important theoretical research value and practical guiding significance. Summary of the Invention

[0005] The first objective of this invention is to provide a strain of Flavobacterium bacteria that can significantly improve the attachment rate and metamorphosis rate of coral larvae. Flavobacteriaceae sp. SCSIO 2518, this bacterium was isolated from calcified algae collected from the sea area of ​​Luhuitou, Sanya, Hainan Province, China. This strain was deposited on November 14, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100, Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC No. 67297.

[0006] Screening experiments on the induction effect of bacteria on coral larvae showed that... Flavobacteriaceae A bacterial solution of sp. SCSIO 2518 fermented for 48 h (concentration 10 μL bacterial solution / mL seawater) significantly promoted the attachment and metamorphosis of coral larvae. After 96 h of induction, the attachment rate of coral larvae was 63.3%, and the metamorphosis rate (the metamorphosis rate mentioned in this article refers to both attached and metamorphosed larvae plus those that did not attach) was 78.3%.

[0007] Through the Flavobacteriaceae Growth curves of sp. SCSIO 2518 were determined and fitted using a logistic model. The results showed that the maximum growth rate of this strain was 0.0497 (OD0.05). 600 The cells (in 23 hours) enter the logarithmic phase and reach the stationary phase at 46 hours. Flow cytometry was used to analyze cells in the logarithmic phase. Flavobacteriaceae sp. SCSIO 2518 is used for counting and compared with the corresponding OD. 600 A linear regression model was established to obtain the relationship between the bacterial density and OD. 600 The corresponding values ​​are: y = 5,210,817,359.51x - 515,470,930.18 R ² = 1.00); where x represents OD. 600 y represents bacterial density (cells / mL).

[0008] By analyzing data from different periods FlavobacteriaceaeThe effect of sp. SCSIO 2518 on inducing coral larval attachment and metamorphosis was verified, and the results showed that the logarithmic phase had the best induction effect. Further analysis was conducted on different bacterial cell concentrations during the logarithmic phase. Flavobacteriaceae The effect of sp. SCSIO 2518 on inducing attachment metamorphosis in coral larvae was verified, and the strain was found to be effective. Flavobacteriaceae The optimal inducing concentration for sp. SCSIO 2518 is 4 μg / L. 7 At this concentration of cells / mL, after 96 h of induction, the attachment rate of coral larvae was 48.3% and the metamorphosis rate was 75%.

[0009] A second objective of this invention is to provide a microbial preparation that promotes attachment metamorphosis in coral larvae, comprising... Flavobacteriaceae sp. SCSIO 2518 cells or their active ingredients can be used in the artificial propagation of corals and the ecological restoration of coral reefs.

[0010] Preferably, the coral larvae are staghorn cup corals ( Pocillopora damicornis )larva.

[0011] Preferably, the aforementioned Flavobacteriaceae sp. SCSIO 2518 cells are in the logarithmic growth phase.

[0012] Further optimization involves placing staghorn cup coral larvae in a solution containing a concentration of 4×10 7 cells / mL Flavobacteriaceae sp. SCSIO 2518 filters seawater.

[0013] The larval density of staghorn cup corals mentioned above is 1 larva / mL of seawater.

[0014] This invention relates to Flavobacterium bacteria isolated and purified from calcified algae collected from the Luhuitou coral reef area in Sanya, Hainan Province, China. Flavobacteriaceae sp. SCSIO 2518, this bacterium can significantly improve the attachment rate and metamorphosis rate of stony coral larvae, shorten the time, and has no toxic side effects. Therefore, this invention can be applied to promote the attachment and metamorphosis of coral larvae, promote the artificial large-scale breeding of corals, provide high-quality seedling resources for coral population recovery, and thus promote the restoration of degraded coral reef ecosystems.

[0015] Flavobacteriaceae sp. SCSIO 2518 was deposited on November 14, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100, Xianlie Middle Road, Guangzhou, 510070, Guangdong Province, China, with accession number GDMCCNo. 67297. Attached Figure Description

[0016] Figure 1 yesFlavobacteriaceae phylogenetic tree of sp. SCSIO 2518;

[0017] Figure 2 In the screening experiment Flavobacteriaceae Induction effect of sp. SCSIO 2518 bacterial culture;

[0018] Figure 3 yes Flavobacteriaceae Growth curve and logarithmic OD of sp. SCSIO 2518 600 - Linear model of bacterial density;

[0019] Figure 4 Different concentrations Flavobacteriaceae The effects of sp. SCSIO 2518 on attachment metamorphosis of staghorn cup coral larvae. Detailed Implementation

[0020] The following embodiments are specific descriptions of the present invention, but not limitations thereof.

[0021] Example 1: Strains Flavobacteriaceae Separation identification of sp. SCSIO 2518

[0022] Strain isolation: Flavobacteriaceae sp. SCSIO 2518 was isolated in December 2022 from calcified algae collected from the Luhuitou Coral Reef area in Sanya. Porolithon onkodes In this study, the culture medium used was 2216E solid medium (Haibo Biotechnology). After isolation and purification, the samples were frozen at -30°C in 30% glycerol tubes (v / v; 30% glycerol + 70% 2216E liquid medium).

[0023] 16S rRNA sequence identification of the strain: Bacterial DNA was extracted using Chelex 100 chelating resin. Specifically, a single colony of the strain was picked and placed in 100 μL of 10% Chelex 100 solution. After boiling in a water bath for 10 min and centrifuging at 12,000 rpm for 10 min, the supernatant was used as a DNA template for PCR amplification of the 16S rRNA gene. The forward and reverse primers were 27F (5'-AGAGTTTGATCCTGGCTCA-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), respectively. The PCR reaction system consisted of 25 μL of 2×EasyTaq SuperMix, 1 μL each of the forward and reverse primers, 1 μL of DNA template, and 22 μL of ddH2O. The PCR program was as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 50°C annealing for 45 s, 72°C extension for 90 s, 30 cycles, and a final extension at 72°C for 10 min. The size and integrity of the PCR products were verified by agarose gel electrophoresis. The PCR products that met the requirements were sent to Tianyi Huiyuan Biotechnology Co., Ltd. (Guangzhou, Guangdong) for Sanger sequencing. Flavobacteriaceae The 16S rRNA sequence of sp. SCSIO 2518 is shown in SEQ ID NO.1. This was determined by comparison with bacterial 16S rRNA sequences in the EzBioCloud database. Flavobacteriaceae The most similar valid published strain to sp. SCSIO 2518 is Croceivirga thetidis (DJ-13(T), similarity 93.43%), below the general threshold for classifying new bacterial genera (94.50%) (Chun et al., 2018, Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. International Journal of Systematic and Evolutionary Microbiology. 68: 461-466). Further, the top 20 most closely related sequences with the corresponding similarity were obtained from this database, and multiple sequence alignment was performed using MEGA11 software. A phylogenetic tree was constructed using neighbor-joining. [Strain] Flavobacteriaceae The tree construction results of sp. SCSIO 2518 are as follows: Figure 1As shown, it belongs to the phylum Bacteroidetes, class Flavobacteriia, order Flavobacteriales, and family Flavobacteriaceae. Because this bacterium shares less than 94.5% sequence similarity with its closely related strains and is divided into a separate branch on the phylogenetic tree, it is preliminarily identified as a potential new genus within the family Flavobacteriaceae, and is named [name missing]. Flavobacteriaceae sp. SCSIO 2518 was deposited on November 14, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100, Xianlie Middle Road, Guangzhou, 510070, Guangdong Province, China, with accession number GDMCC No. 67297.

[0024] Example 2: Strains Flavobacteriaceae Preliminary determination of the induction effect of sp. SCSIO 2518

[0025] I. Strains Flavobacteriaceae SP. SCSIO 2518 Activation

[0026] Remove the strain from the preservation tube, thaw at room temperature, and spread 50 μL evenly on 2216E solid medium (Haibo Biotechnology). Incubate at 28°C for 3 days in a constant temperature incubator, and then select a single colony for streak purification.

[0027] II. Strains Flavobacteriaceae Preliminary determination of the induction effect of sp. SCSIO 2518

[0028] A loopful of purified bacteria was inoculated into 30 mL of 2216E liquid medium and fermented at 28°C and 180 rpm for 48 h to obtain the bacterial broth (bacterial suspension). The induction effect of this broth on the attachment metamorphosis of staghorn cup coral larvae was investigated. The attachment experiment was conducted in sterile six-well plates. The experimental group consisted of 10 coral larvae + 9.9 mL of sterile filtered seawater (AFSW) + 100 μL of bacterial suspension; the blank control group consisted of 10 coral larvae + 9.9 mL of sterile filtered seawater (AFSW) + 100 μL of 2216E liquid medium. Each treatment was repeated in six replicates. After 48 h or 96 h, the attachment metamorphosis and mortality of the larvae were observed and recorded under a dissecting microscope. The results are as follows: Figure 2 As shown, after 48 h, the attachment rate of staghorn cup coral larvae was 31.7% and the metamorphosis rate was 36.7%; after 96 h, the attachment rate was 63.3% and the metamorphosis rate was 78.3%, which were significantly higher than the blank control, indicating that it has the potential to effectively promote the attachment and metamorphosis of coral larvae.

[0029] Example 3: Strains Flavobacteriaceae Growth curve determination of sp. SCSIO 2518

[0030] I. Strains

[0031] Flavobacteriaceae Growth curve determination of sp. SCSIO 2518

[0032] One loopful of activated and purified bacteria was inoculated into 30 mL of 2216E liquid medium and fermented at 28°C and 180 rpm for 24 h to prepare a seed culture. A 1:100 v / v inoculation volume (500 μL) was then inoculated into 50 mL of fresh 2216E liquid medium (n = 3) and cultured with shaking at 28°C and 180 rpm. Samples were taken from the three culture flasks at predetermined time points, and the OD of the bacterial suspension was measured using a microplate reader. 600 Value (n = 3), with sterile 2216E medium as a blank control, strain Flavobacteriaceae sp. The SCSIO 2518 measurement cycle is 72 hours; using the logistic model. Perform fitting, where Asym For the maximum OD value, Xmid The inflection point time of the logarithmic period. Scal The scaling parameter (negatively correlated with curve steepness) is obtained through the formula... μ max = Asym / (4× Scal Calculate the maximum growth rate (OD) 600 / h), based on the empirical formula Lag phase ≈ Xmid - 2× Scal Estimate the lag phase. (Bacterial strain) Flavobacteriaceae The growth curve of sp. SCSIO 2518 is as follows: Figure 3 As shown in Figure A, the fitted curve is... Based on estimation, the strain Flavobacteriaceae The hysteresis period of sp. SCSIO 2518 was 23 hours (after which it entered the growth phase), and it gradually entered the stationary phase at 46 hours, with a maximum growth rate of 0.0497 (OD). 600 / h).

[0033] II. Strains Flavobacteriaceae sp. SCSIO 2518 logarithmic phase cell density - OD 600 Linear model drawing

[0034] At the sampling time points of the above growth curve measurement, an additional 1.96 mL of bacterial suspension was taken for bacterial density quantification (cells / mL). The steps are as follows: (1) Bacterial suspension fixation: Add 40 μL of glutaraldehyde, fix the sample for 5 minutes, freeze in liquid nitrogen and store at -30°C. (2) Cell counting: Take out the logarithmic phase sample of the strain, thaw at room temperature, take 1 mL and filter it through a 600-mesh sieve, dilute the filtrate to an appropriate concentration with sterile ddH2O, add 10 μL of SYBR staining solution, stain in the dark for 10 minutes, and then count the sample using flow cytometry. Select the OD at the logarithmic phase time points (25-35 h, 38-39 h). 600 A linear regression model was established between the values ​​and the corresponding cell densities, and the results are as follows: Figure 3 As shown in B. (Strain) Flavobacteriaceae Logarithmic phase bacterial density and OD of sp. SCSIO 2518 600 The corresponding values ​​are y = 5,210,817,359.51x - 515,470,930.18 ( R ² = 1.00); where x represents OD. 600 y represents bacterial density (cells / mL).

[0035] Example 4: Identification of the bacterial strain Flavobacteriaceae sp.SCSIO 2518 optimal induction concentration

[0036] The staghorn cup coral was collected in August 2024 from the waters of Luhuitou, Sanya, Hainan Province, China. After the larvae were collected, they were cultured indoors in sand-filtered seawater at 27°C for later use.

[0037] Based on the growth curve, take the strain Flavobacteriaceae sp. SCSIO 2518 in logarithmic growth phase suspension was centrifuged at 8000 rpm for 10 min at room temperature. After discarding the supernatant, the suspension was resuspended in sterile filtered seawater (AFSW) and diluted to 1×10⁻⁶. 7 2×10 7 4×10 7 6×10 7 8×10 7 1×10 8 Larval attachment experiments were conducted using cells / mL. The attachment experiments were performed in sterile six-well plates. The experimental group consisted of 10 coral larvae + 10 mL AFSW + corresponding gradient concentrations of bacteria; the blank control group consisted of 10 coral larvae + 10 mL AFSW. Each treatment was repeated 6 times. The experimental light intensity was approximately 70 μmol photons / mL. -2 s -1The light / dark cycle was 12 h / 12 ​​h, and the temperature was controlled at 27±1°C. During this period, larval attachment, metamorphosis, and death were observed and recorded under a dissecting microscope at 48 h and 96 h; half of the seawater and bacterial cells in the system were replaced at 48 h. (Bacterial strains...) Flavobacteriaceae The induction experiment results of sp.SCSIO 2518 are as follows: Figure 4 As shown. After 48 h, the adhesion rate of the strain-treated groups ranged from 0% to 16.7%, and the metamorphosis rate ranged from 0% to 45%, with no statistically significant difference compared to the control group. However, after 96 h, the adhesion rate of the strain-treated groups ranged from 1.7% to 48.3%, and the metamorphosis rate ranged from 3.3% to 75%, of which 4×10 7 and 8×10 7 The metamorphosis rates in the cell / mL concentration groups were 75% and 66.7%, respectively, significantly higher than those in the control group, and no larvae died in either group after 96 h. In conclusion, the optimal inducing concentration of this bacterium is 4 × 10⁻⁶ cells / mL. 7 cells / mL.

[0038] The above are merely preferred embodiments of the present invention. It should be noted that the above embodiments do not limit the present invention. Any technical solutions obtained by optimization and improvement based on the above solutions do not exceed the protection scope of the present invention.

[0039] Flavobacteriaceae The 16S rRNA sequence of sp. SCSIO 2518 (SEQ ID NO.1)

[0040]

Claims

1. Flavobacteriaceae Flavobacteriaceae sp. SCSIO 2518, accession number GDMCC No. 67297.

2. A type of coral that can promote the growth of staghorn cup-shaped corals ( Pocillopora damicornis A microbial preparation for larval attachment metamorphosis, characterized in that, Containing the contents of claim 1 Flavobacteriaceae sp. SCSIO 2518 cells or culture.

3. The Flavobacterium family bacteria as described in claim 1 Flavobacteriaceae The use of the microbial preparations described in sp. SCSIO 2518 or claim 2 in promoting attachment metamorphosis of staghorn cup coral larvae.

4. The application according to claim 3, characterized in that, The aforementioned Flavobacteriaceae sp. SCSIO2518 is a logarithmic growth phase cell.

5. The application according to claim 3, characterized in that, Staghorn cup coral larvae were placed in a solution with a concentration of 4×10 7 cells / mL Flavobacteriaceae sp. SCSIO 2518 filters seawater.

6. The application according to claim 3, characterized in that, The density of staghorn cup coral larvae mentioned above is 1 larva / mL of seawater.

Images