A method for preventing and controlling harmful algae in water bodies using sphingosine substances

Abstract

The invention discloses a method for preventing and controlling harmful algae in water bodies by using sphingosine substances, adding sphingosine substances to water bodies where algae are about to break out or have already broken out, and selecting sphingosine substances of different types and concentrations for different control objects, so as to achieve the best control effect. The method of the invention has the advantages of natural source, simple operation, obvious effect, and small secondary pollution.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental protection, and in particular relates to a method for preventing and controlling harmful algae in water bodies by utilizing sphingosine substances. Background Art

[0002] Harmful algae refer to algae that reproduce rapidly in water bodies, consume a large amount of nutrients and oxygen in the water body, produce unpleasant odors on the water surface, and even release toxins into the water environment. In fresh water, common harmful algae in my country's rivers, lakes and reservoirs include Microcystis, Chlorella, Anabaena, Oscillatoria, Aphanizomenon, etc.; in seawater, harmful algae that frequently erupt in my country's waters include Amphidiniumcarterae, Phaeocystis globosa, Heterosigma akashiwo, and Skeletonema costatum, etc. Harmful algae outbreaks are major water safety accidents that damage the health of aquatic ecosystems, threaten public drinking water safety, and affect regional economies and ecological civilizations.

[0003] Suitable water temperature, light and other environmental conditions are accidental factors that induce harmful algae outbreaks. The disorder of water ecological structure and eutrophication of water bodies are the root causes of harmful algae outbreaks. The development of social economy has led to a large amount of land nutrients being discharged into water bodies. Eutrophication of water bodies is the basic status of water bodies in my country at present and for a long time to come. Therefore, the development of efficient, environmentally friendly, green and friendly new algae control agents is an important topic in the field of harmful algae prevention and control.

[0004] According to the source, algaecides can be divided into two categories: artificial synthesis and natural sources. Common synthetic algaecides include broad-spectrum algaecides such as potassium permanganate, hydrogen peroxide and copper sulfate. They have good algaecidal effects, but poor controllability, and often cause damage to some non-target organisms such as fish, crustaceans, benthic organisms, etc. Natural algaecides are mainly isolated from terrestrial or aquatic plants and microorganisms. They are mainly composed of secondary metabolites such as phenolic acid and its derivatives, fatty acids and their derivatives, amino acids, alkaloids, terpenes, etc., and have the characteristics of strong specificity, good effect, and small side effects. Among them, the types of plant secondary metabolites are complex and varied, and their properties vary. Therefore, it is very necessary to continuously explore and develop new and efficient algaecides for the prevention and control of harmful algae. Summary of the invention

[0005] The technical problem to be solved by the present invention is to find a safe, environmentally friendly, and target-specific efficient novel algaecide to solve the problem of frequent occurrence of harmful algae in water bodies.

[0006] To achieve the above object, the present invention is implemented by the following technical scheme: a method for preventing and controlling harmful algae in water bodies, which is implemented by adding sphingosine substances to water bodies where algae are about to break out or have already broken out. Different types and concentrations of sphingosine substances are selected for different control objects to achieve the best control effect.

[0007] Furthermore, the control targets of harmful algae include blue algae and green algae blooms caused by Microcystis aeruginosa and Chlorella in freshwater, and red tides caused by Amphidinium carterae, Phaeocystis globosa, Heterosigma akashiwo, Isochrysis galbana, and Skeletonema costatum in seawater.

[0008] Furthermore, the sphingosine substances include 4-hydroxysphingosine (phytosphingosine, PHS), D-sphingosine (sphingosine, SPH), D-dihydrosphingosine (sphinganine, DHS) and N-acetyl-D-sphingosine (N-acetylsphingosine, APH), as well as other derivatives with sphingosine as the basic skeleton.

[0009] Furthermore, D-dihydrosphingosine was used to control red tides dominated by Amphidinium carterae.

[0010] Furthermore, 4-hydroxysphingosine is used to control red tides in which Heterosigma akashiwo, Phaeocystis globosa, Isochrysis galbana or Skeletonema costatum are dominant species.

[0011] Furthermore, 4-hydroxysphingosine is used to control algal blooms in which Microcystis aeruginosa or Chlorella vulgaris is the dominant species.

[0012] Furthermore, the forms of sphingosine substances include but are not limited to powders and liquid medicines, and the methods of use include but are not limited to directly spraying solutions, hanging sustained-release bags, administering slow-release particles, and the like.

[0013] The beneficial effects of the present invention are:

[0014] 1. The sphingosine substances used in the present invention are a class of aliphatic compounds with a skeleton of 18 carbon atoms, one amino group and two hydroxyl groups at C-2, C-1 and C-3 respectively, belonging to sphingolipids, and are components of cell membranes. Sphingosine can be found in lipid-rich organs of animals, plants and fungi, has a natural source, and is highly safe to use.

[0015] 2. The sphingosine substances used in the present invention have different inhibitory effects on different algae. In seawater, sphingosine substances can effectively inhibit the growth of common harmful algae in my country's sea areas; in freshwater bodies, relatively low concentrations of sphingosine substances can effectively control the growth of blue algae. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The fitting curves of the effective inhibitory concentrations of 4-hydroxysphingosine (A), D-sphingosine (B), D-dihydrosphingosine (C) and N-acetyl-D-sphingosine (D) on A. carterae at 72 h;

[0017] Figure 2 The fitting curves of the effective inhibitory concentrations of 4-hydroxysphingosine (A), D-sphingosine (B), D-dihydrosphingosine (C) and N-acetyl-D-sphingosine (D) on H. akashiwo at 72 h;

[0018] Figure 3 The fitting curves of the effective inhibitory concentrations of 4-hydroxysphingosine (A), D-sphingosine (B), D-dihydrosphingosine (C) and N-acetyl-D-sphingosine (D) on P. globosa at 72 h;

[0019] Figure 4 The fitting curves of the effective inhibitory concentrations of 4-hydroxysphingosine, D-sphingosine and D-dihydrosphingosine on Microcystis aeruginosa FACHB905 (A, B, C) and Chlorella vulgaris (D, E, F) at 72 h, respectively;

[0020] Figure 5 The fitting curves of the effective inhibitory concentrations of 4-hydroxysphingosine, D-sphingosine and D-dihydrosphingosine on Isochrysis galbana (A, B, C) and S. costatum (D, E, F) at 72 h, respectively;

[0021] Figure 6This is the fitting curve of the effective inhibitory concentration of D-dihydrosphingosine on A.carterae at 120h;

[0022] Figure 7 The effect of D-dihydrosphingosine on the physiological activities (AF) and gene expression of A. carterae cells at the half effective inhibitory concentration of 120 h (G), the dotted line in G is the expression level of the control group;

[0023] Figure 8 The molecular docking results of D-dihydrosphingosine on proteins related to A.carterae. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

[0025] Example 1: Test of the anti-algae activity of sphingosines against A. carterae

[0026] Using Amphidinium robustum as the test object, the initial density was 5×10 4 10 μL of a series of concentration gradients of 4-hydroxysphingosine (PHS), D-sphingosine (SPH), D-dihydrosphingosine (DHS) and N-acetyl-D-sphingosine (APH) were added to Amphidinium robustum cells / mL in the logarithmic growth phase, and the same dose of dimethyl sulfoxide (DMSO) was added as a control.

[0027] After 72 hours of culture, the inhibition rate under each test concentration was calculated using the following formula:

[0028] Inhibition rate IR (%) = (D Spi -D DMSO ) / D DMSO ×100%

[0029] Where D Spi is the algal cell density of the sphingosine-treated group, D DMSO is the cell density of the DMSO control group. The inhibitory effects of the four sphingosines at different test concentrations are shown in Table 1.

[0030] Table 1 Effects of different sphingosine concentrations on the growth of Amphidinium robustum

[0031]

[0032] The inhibition rate was fitted according to the probit regression model, and the half effective inhibition concentration (EC 50 ), the test results are shown in Table 2, and the inhibition rate fitting curve is shown in Figure 1 , r 2 It is the overall fit of the regression equation of inhibition rate and test concentration. Preferably, 0.224 mg / L of D-dihydrosphingosine is used to control red tide with Amphidinium carterae as the dominant species.

[0033] Table 2 The effective inhibitory concentration of sphingosines against Amphidinium robustum for 72 hours

[0034]

[0035] Example 2: Test of the anti-algae activity of sphingosines against H. akashiwo

[0036] Using Heterosigma akashiwo as the test object, the initial density was 5×10 4 10 μL of a series of concentration gradients of 4-hydroxysphingosine (PHS), D-sphingosine (SPH), D-dihydrosphingosine (DHS) and N-acetyl-D-sphingosine (APH) were added to Heterosigma akashiwo in the logarithmic growth phase, and the same dose of dimethyl sulfoxide was added as a control. After 72 hours of culture, the inhibition rate under each test concentration condition was calculated according to the method of Example 1. The inhibitory effects of different test concentrations of the four sphingosines are shown in Table 3.

[0037] Table 3 Effects of different sphingosine concentrations on the growth of Heterosigma akashiwo

[0038]

[0039] The inhibition rate was fitted according to the probit regression model, and the half effective inhibition concentration (EC 50 ), the test results are shown in Table 4, and the inhibition rate fitting curve is shown in Figure 2 Preferably, 0.066 mg / L of 4-hydroxysphingosine is used to control red tides dominated by Heterosigma akashiwo.

[0040] Table 4 The effective inhibitory concentration of sphingosines against Heterosigma akashiwo for 72 hours

[0041]

[0042] Example 3: Test of the anti-algae activity of sphingosines against P. globosa

[0043] The spherical brown cyst was used as the test object, and the initial density was 5×10 5 10 μL of a series of concentration gradients of 4-hydroxysphingosine (PHS), D-sphingosine (SPH), D-dihydrosphingosine (DHS) and N-acetyl-D-sphingosine (APH) were added to P. sphericalus cells / mL in the logarithmic growth phase, and the same dose of dimethyl sulfoxide was added as a control. After 72 hours of culture, the inhibition rate under each test concentration condition was calculated according to the method of Example 1. The inhibitory effects of different test concentrations of the four sphingosines are shown in Table 5.

[0044] Table 5 Effects of different sphingosine concentrations on the growth of Phaeocystis sphericalensis

[0045]

[0046] The inhibition rate was fitted according to the probit regression model, and the half effective inhibition concentration (EC 50 ), the test results are shown in Table 6, and the inhibition rate fitting curve is shown in Figure 3 Preferably, 0.060 mg / L of 4-hydroxysphingosine is used to control red tides dominated by Phaeocystis globosa.

[0047] Table 6 The effective inhibitory concentration of sphingosines against Pseudocystis sphericalensis for 72 hours

[0048]

[0049] Example 4: Test of the anti-algae activity of sphingosines against Microcystis aeruginosa (M. aeruginosa FACHB905)

[0050] Microcystis aeruginosa was used as the test object. The initial density was 5×10 6 10 μL of a series of concentration gradients of 4-hydroxysphingosine (PHS), D-sphingosine (SPH), and D-dihydrosphingosine (DHS) were added to Microcystis aeruginosa cells / mL in the logarithmic growth phase, and the same dose of dimethyl sulfoxide was added as a control. After 72 hours of culture, the inhibition rate under each test concentration condition was calculated according to the method of Example 1. The inhibition effects of different test concentrations of the three sphingosines are shown in Table 7.

[0051] Table 7 Effects of different sphingosine concentrations on the growth of Microcystis aeruginosa

[0052]

[0053]

[0054] The inhibition rate was fitted according to the probit regression model, and the half effective inhibition concentration (EC 50 ), the test results are shown in Table 8, and the inhibition rate fitting curve is shown in Figure 4 Preferably, 0.028 mg / L of 4-hydroxysphingosine is used to control algal blooms in which Microcystis aeruginosa is the dominant species.

[0055] Table 8 The effective inhibitory concentration of sphingosines against Microcystis aeruginosa for 72 hours

[0056]

[0057] Example 5: Test of the anti-algae activity of sphingosines against Chlorella vulgaris

[0058] Chlorella vulgaris was used as the test object, and the initial density was 5×10 5 10 μL of a series of concentration gradients of 4-hydroxysphingosine (PHS), D-sphingosine (SPH), and D-dihydrosphingosine (DHS) were added to Chlorella cells / mL in the logarithmic growth phase, and the same dose of dimethyl sulfoxide was added as a control. After 72 hours of culture, the inhibition rate under each test concentration condition was calculated according to the method of Example 1. The inhibitory effects of different test concentrations of the three sphingosines are shown in Table 9.

[0059] Table 9 Effect of different sphingosine concentrations on the growth of Chlorella

[0060]

[0061] The inhibition rate was fitted according to the probit regression model, and the half effective inhibition concentration (EC 50 ), the test results are shown in Table 10, and the inhibition rate fitting curve is shown in Figure 4 Preferably, 0.936 mg / L of 4-hydroxysphingosine is used to control blooms in which Chlorella vulgaris is the dominant species.

[0062] Table 10 The effective inhibitory concentration of sphingosine substances on Chlorella for 72h

[0063]

[0064] Example 6: Test of the anti-algae activity of sphingosines against Isochrysis galbana

[0065] Isochrysis galbana was used as the test object. The initial density was 5×10 5 10 μL of a series of concentration gradients of 4-hydroxysphingosine (PHS), D-sphingosine (SPH), and D-dihydrosphingosine (DHS) were added to Isochrysis galbana cells / mL in the logarithmic growth phase, and the same dose of dimethyl sulfoxide was added as a control. After 72 hours of culture, the inhibition rate under each test concentration condition was calculated according to the method in Example 1. The inhibitory effects of different test concentrations of the three sphingosines are shown in Table 11.

[0066] Table 11 Effects of different sphingosine concentrations on the growth of Isochrysis galbana

[0067]

[0068] The inhibition rate was fitted according to the probit regression model, and the half effective inhibition concentration (EC 50 ), the test results are shown in Table 12, and the inhibition rate fitting curve is shown in Figure 5 Preferably, 0.008 mg / L of 4-hydroxysphingosine is used to control red tides dominated by Isochrysis galbana.

[0069] Table 12 The effective inhibitory concentration of sphingosines on Isochrysis galbana for 72h

[0070]

[0071] Example 7: Test of the anti-algae activity of sphingosines against S. costatum

[0072] Using Skeletonema costatum as the test object, the initial density was 5×10 4 10 μL of a series of concentration gradients of 4-hydroxysphingosine (PHS), D-sphingosine (SPH), and D-dihydrosphingosine (DHS) were added to Skeletonella costatum cells / mL in the logarithmic growth phase, and the same dose of dimethyl sulfoxide was added as a control. After 72 hours of culture, the inhibition rate under each test concentration condition was calculated according to the method of Example 1. The inhibitory effects of different test concentrations of the three sphingosines are shown in Table 13.

[0073] Table 13 Effects of different sphingosine concentrations on the growth of Skeletonema costatum

[0074]

[0075] The inhibition rate was fitted according to the probit regression model, and the half effective inhibition concentration (EC 50), the test results are shown in Table 14, and the inhibition rate fitting curve is shown in Figure 5 Preferably, 0.01 mg / L of 4-hydroxysphingosine is used to control red tides dominated by Skeletonema costatum.

[0076] Table 14 The effective inhibitory concentration of sphingosines on Skeletonema costatum for 72 hours

[0077]

[0078] Example 8: Study on the inhibitory mechanism of D-dihydrosphingosine on A.carterae

[0079] 1 Materials and methods

[0080] 1.1 Algal strains and reagents

[0081] A. carterae was provided by the Institute of Marine Biology, College of Oceanography, Zhejiang University. D-dihydrosphingosine (DHS) was purchased from J&K Scientific, and dimethyl sulfoxide (DMSO) was purchased from Aladdin.

[0082] 1.2 Anti-algae activity test

[0083] During the activity test, the initial density of 100 mL was 5×10 4 cells / mL of Amphidinium robustum was added with 10 μL of a series of concentration gradients of DHS, and 10 μL of dimethyl sulfoxide was added as a control. After the 5th day, the algal cells of the treatment group and the control group with DHS and dimethyl sulfoxide added were sampled and fixed, counted using a hemocytometer, and the inhibition rate at each drug concentration was calculated. Finally, the curve was fitted according to the probability unit regression model (Probit regression) to calculate the half effective inhibition concentration of DHS on the 5th day.

[0084] 1.3 Determination of biochemical and physiological indicators

[0085] When studying the mechanism of DHS inhibition of algae, in order to obtain sufficient algal cell mass, the half effective inhibition concentration (EC 5d-50 =0.13 mg / L), for an initial density of 5×10 4 cells / mL of Amphidinium robustum for 5 days.

[0086] Determination of chlorophyll fluorescence parameters: A portable modulated chlorophyll fluorescence instrument was used to measure the chlorophyll fluorescence parameters of the treatment group. Before the measurement, the collected algae cells were first allowed to dark adapt for 30 minutes, and then the maximum photochemical quantum yield (Fv / Fm) of the algae cell photosystem II (PSⅡ), the maximum relative electron transfer rate (rETR max ) and the initial slope (α) of the light response curve that characterizes the light energy utilization efficiency.

[0087] Determination of reactive oxygen free radicals (ROS): The fluorescent probe DCFH-DA was used to measure the reactive oxygen species in the cells. During the measurement, freshly treated algal cells were collected and first incubated with 10 μM DCFH-DA (37°C, 20 min). After the incubation, the fluorescent dye was washed with a phosphate buffer (pH 7.8, 50 mM) without the probe. After washing three times, the fluorescence value of DCF in the cells was measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0088] Collection of algal cells for other physiological indicators: The robust algae treated with DHS for 5 days were used as the collection object, and 100 mL of the treated algal cells were collected using a desktop refrigerated high-speed centrifuge (3500 rpm, 10 min, 4°C), and the collected algal cells were frozen with liquid nitrogen and finally stored at -80°C for later use.

[0089] Determination of enzyme activity and malondialdehyde (MDA): Algal cell disruption: After the frozen algal cells were taken out, the disruption process was completed on ice. When disrupting, 2 mL of phosphate buffer was first added to the thawed cells, and after the algal cells were resuspended, they were disrupted using an ultrasonic disruptor (3 min, 5 s, 5 s). After the disrupted algal cells were subjected to frozen centrifugation (10000 rpm, 10 min, 4 ° C), the supernatant was taken and stored at -80 ° C for later use.

[0090] The total protein content of algal cells was determined by the Coomassie brilliant blue method; the superoxide dismutase (SOD) activity was determined by the hydroxylamine method; the catalase (CAT) activity was determined by the ammonium molybdate method; and the MDA content was determined by the thiobarbituric acid (TBA) method.

[0091] 1.4 Gene expression analysis

[0092] Real-time fluorescence quantitative PCR (qPCR) was used to analyze the relative expression of key genes. Before the measurement, freshly treated algal cells with DHS were first collected and frozen in liquid nitrogen. The total RNA of algal cells was then extracted using the TaKaRa total RNA extraction kit (NO.9769) and the purity of the RNA (OD 260 / 280) was greater than 2.0. Then, the cDNA sample required for qPCR was obtained using TaKaRa reverse transcription kit (NO.RR036A).

[0093] During the measurement, 18S rRNA with relatively stable expression level was used as the internal reference gene. The 20μL qPCR reaction system included 10μL Green qPCR SuperMix, 0.8 μL of forward and reverse primers (10 μM), 2 μL of cDNA template and 6.4 μL of sterile water. The amplification program was: pre-denaturation, 95°C for 10 min; amplification (40 cycles), 95°C for 15 s, 60°C for 20 s; melting curve, 95°C for 15 s, 60°C for 1 min. The relative expression level of the target gene was calculated according to the following formula: relative expression level = 2 -ΔΔCt , where -ΔΔCt=(Ct rt -Ct rc )-(Ct tt -Ct tc ), rt represents the Ct value of the reference gene in the treatment group, rc represents the Ct value of the reference gene in the control group, tt represents the Ct value of the target gene in the treatment group, and tc represents the Ct value of the target gene in the control group.

[0094] 1.5 Molecular docking

[0095] To further study the binding ability of DHS with key proteins, the molecular docking method was used to study the interaction between drugs and proteins. Before molecular docking, the protein homology structure was first constructed using protein sequence and homology modeling software. The three-dimensional crystal structure of DHS was downloaded from the Pubchem database by CAS number (764-22-7). Then, all possible binding pockets of protein receptors were predicted by the online server pocasa, and the parameters of the binding pocket were determined using the GetBox Plugin plug-in in the PyMOL software. Finally, the most stable conformation was selected and the hydrogen bonds and hydrophobic interactions between the protein-ligand complex were analyzed using Ledock and Ligplot software respectively.

[0096] 1.6 Experimental data analysis

[0097] SPSS 22.0 was used to perform independent sample T test statistical analysis and probability unit regression analysis on the data. P < 0.05 was considered a significant difference (*), and p < 0.01 and p < 0.001 were considered extremely significant differences (**, ***).

[0098] 2 Results

[0099] 2.1 The effective inhibitory concentration (EC) of DHS on Amphidinium robustum on the 5th day 50 )

[0100] Depend on Figure 6 It can be seen that the treatment of Amphidinium robustum with 0.05 mg / L, 0.10 mg / L, 0.15 mg / L, 0.20 mg / L and 0.30 mg / L DHS respectively can effectively inhibit the growth of Amphidinium robustum compared with the control group with dimethyl sulfoxide (see Table 15). Through the probability unit model regression analysis, the regression formula of D-dihydrosphingosine inhibition rate y (%) and test concentration x (mg / L) is y=1.738+1.992logx(r 2 =0.972), the model predicted that the effective inhibitory concentration of D-dihydrosphingosine on Amphidinium robustum on the 5th day was 0.13 mg / L. In the verification experiment, this dose of drug was added to Amphidinium robustum, and the algal cell density of the treatment group was measured to be 23.2×10 4 cells / mL, and the cell density of the control group was 46.0×10 4 cells / mL, and the inhibition rate was 49.57%.

[0101] Table 15 Inhibitory effect of different concentrations of DHS on the growth of Amphidinium robustum at 120 h

[0102]

[0103] 2.2 Effects of DHS on the photosynthetic system of Amphidinium robustum

[0104] Depend on Figure 7 As shown in A and B, DHS at the half effective inhibitory concentration on the 5th day can significantly inhibit the maximum photochemical quantum yield and light energy utilization efficiency of PSⅡ of M. robusta. Compared with the control group, Fv / Fm and α decreased by 20.27% (p<0.01) and 21.24% (p<0.01), respectively, while the maximum relative electron transfer rate characterizing the maximum photosynthetic rate decreased slightly, but the difference was not significant (not shown in the figure).

[0105] 2.3 Effects of DHS on the antioxidant system of Amphidinium robustum

[0106] Depend on Figure 7 As shown in Figure C, after DHS treatment, the DCF fluorescence intensity, which represents the level of intracellular reactive oxygen species in Amphidinium robustum, increased by 1.73 times compared with the control group, and there was a significant difference between the two (p<0.01). Figure 7 As shown in Figures D and E, compared with the control group, the intracellular SOD and CAT activities of Amphidinium robustum were significantly increased after treatment, indicating that the reactive oxygen species induced by DHS greatly activated the scavenging ability of the intracellular antioxidant system. Figure 7The results in Figure F show that compared with the control group, the MDA content, which represents the level of membrane lipid peroxidation, increased by 41.33% after DHS treatment (p<0.001), indicating that membrane lipid peroxidation occurred in the cell membrane of M. robusta.

[0107] 2.4 Effects of DHS on the expression of key genes in Amphidinium robustum

[0108] Depend on Figure 7 The results of middle G showed that after DHS treatment, the gene expression levels of proteins involved in nitrate (HANT), nitrite (NIT), phosphate (PST) transport and organic phosphorus (ALP) utilization in Amphidinium robustum were significantly upregulated, among which high affinity phosphate transporter and nitrite transporter were upregulated by 3.24 (p<0.01) and 3.29 (p<0.01) times, respectively, while the expression level of ammonium transporter involved in ammonium (AMT) transport was not significantly different from that in the control group. In the photosynthetic system, the expression level of the gene encoding apolipoprotein A1 (Psa A) of photosystem I (PSⅠ) in the DHS treatment group was upregulated by 73.30% (p<0.01), the expression levels of PSⅠ core protein (Psa B) and PSⅡD1 protein (Psb A) did not change significantly, while the expression level of ribulose-1,5-bisphosphate carboxylase / oxygenase II (RuB) involved in the Calvin cycle decreased by 47.60% (p<0.001). In addition, the expression of rhodopsin (RHO) that produces ATP by capturing light energy, heat shock protein 90 (HSP90) involved in cell environmental defense, and arginine / lysine-specific cysteine ​​protease (MET) involved in cell apoptosis were also significantly upregulated, increasing by 1.67 (p<0.001), 2.14 (p<0.01) and 2.33 (p<0.01) times, respectively.

[0109] 2.5 Docking of DHS with key protein molecules of Amphidinium robustum

[0110] In order to study the binding ability of DHS with differentially expressed gene proteins of Amphidinium robustum, molecular docking was used to further study the interaction between the drug and different receptors. According to the size of the binding energy predicted by molecular docking, the affinity of DHS ligand to each receptor is as follows: RuB (-6.82 kcal / mol) > RHO (-6.76 kcal / mol) > MET (-6.74 kcal / mol) > HANT (-6.01 kcal / mol) = Psa A (-6.01 kcal / mol) > HSP90 (-5.81 kcal / mol) > ALP (-5.59 kcal / mol) > NIT (-5.56 kcal / mol) > Pet A (-5.18 kcal / mol). Figure 8As shown in A, there are 5 hydrogen bonds in the DHS-RuB complex. The amino acid residues that form the structure include Asp550 and Asp553. The 12 hydrophobic bonds are formed by His504, Arg505, Ala506, His508, His509, Thr512, Thr521, Ala522, Thr539, Met547, Glu548, and Gly549. Figure 8 As shown in Figure B, in the DHS-RHO complex, DHS forms 4 hydrogen bonds with Glu97 and Ala121, and 11 hydrophobic bonds with Met27, Met29, Val58, Arg70, Gln87, Phe96, Trp99, Glu123, Met124, Ile136, and Ile152. Figure 8 As shown in C, in the DHS-MET complex, six amino acid residues, Cys22, Asp25, Ser83, Gly84, Asp136, and Cys138, are involved in the formation of nine hydrogen bonds, and Asn24, Asn28, Cys137, Gly191, Gly192, and Asn196 form six hydrophobic bonds, respectively.

[0111] 3. Discussion

[0112] 3.1 Inhibitory effect of DHS on Amphidinium robustum

[0113] According to the drug concentration gradient test and the prediction results of the probability unit regression model, the half effective inhibitory dose of DHS for Amphidinium robustum for 120 hours is 0.13 mg / L (0.44 μM), and the actual inhibition efficiency of the theoretical concentration of the half inhibitory dose is 49.57%, which proves that the inhibitory effect of DHS on Amphidinium robustum is stable and the prediction results of the probability regression model are reliable. The DHS studied in this embodiment is a new and efficient natural allelopathic substance, and its control effect on common algae blooms and red tide algae is better than the vast majority of natural allelopathic substances that have been identified so far. For example, phenolic acids and fatty acids are common allelopathic substances released by large aquatic plants in water bodies. Among them, catechol and ethyl 2-methylacetoacetate, which have outstanding algae inhibition properties, have half effective inhibition concentrations of 2.72 μM for Microcystis aeruginosa, respectively. [1] and 4.51 μM [2] Alkaloids are another common allelopathic substance secreted by plants. The median effective concentration of alkaloids (3-(dimethylaminomethyl)indole) isolated and extracted from Phragmites australis is 12.05 μM against Microcystis aeruginosa. [3]In recent years, the anti-algae effect of natural flavonoids has been discovered one after another. Huang Haomin, Li Chao and others have conducted in-depth and systematic research on the anti-algae activity-structure relationship of this type of substance and found that the 5'4-dihydroxyflavone with the strongest allelopathic effect has a half effective inhibition concentration of 1.85μM for Microcystis aeruginosa and Phaeocystis sphericalensis. [4] and 2.70 μM [5] According to the test results of Example 3 and Example 4, the half effective inhibition concentrations of DHS against Phaeocystis spherical and Microcystis aeruginosa were 0.21 μM (0.062 mg / L) and 0.31 μM (0.094 mg / L), respectively. The effect of the new allelopathic substance studied in this example is one order of magnitude higher than that of the common allelopathic substances.

[0114] 3.2 Effects of DHS on the photosynthetic and antioxidant systems of Amphidinium robustum

[0115] In this example, the chlorophyll fluorescence parameters of DHS-treated Dichloa robusta were measured in situ, and it was found that DHS mainly affects the photosynthesis of algae by affecting the light energy utilization efficiency of algae and destroying the transmission of PSⅡ electron chain. Photosynthesis is an important link in which algae synthesize water and carbon dioxide into the energy necessary for life activities by absorbing light energy, passing through the light reaction and dark reaction stages. The light reaction occurs on the thylakoid membrane of the chloroplast, and mainly includes four steps: light energy absorption, water photolysis, electron transfer and light energy conversion. The absorption of light and electron transfer in the light reaction are blocked, which will affect the synthesis of ATP and nicotinamide adenine dinucleotide phosphate (NADPH), making it impossible for the light reaction to provide reducing coenzymes and energy for the dark reaction, thereby further affecting the synthesis of organic matter in the dark reaction, and ultimately limiting the reproduction and growth of algae.

[0116] Reactive oxygen is an important signaling molecule in the growth and metabolism of organisms. It is involved in cell growth and reproduction, defense response, morphology construction and cell death, and can regulate the expression of corresponding genes. For algal cells under normal conditions, the complete antioxidant system in the cells can keep the production and consumption of intracellular reactive oxygen in a dynamic equilibrium state. However, when the body encounters external environmental stress, reactive oxygen molecules will be produced in large quantities and gradually exceed the clearance limit of the antioxidant system. Excessive reactive oxygen will accumulate in the cell, produce oxidative stress, damage intracellular biomacromolecules such as DNA and protein, and further destroy the structure and normal physiological function of other organelles, ultimately leading to cell death. The addition of DHS in this example induced the massive production of reactive oxygen, so that the intracellular ROS level of the strong angiospermum treatment group was significantly higher than that of the control group. As the first line of defense of the antioxidant system, ROS will first Disproportionately disproportionately disproportionately disproportionately disproportionately into H2O2 and O2, after which CAT further catalyzes H2O2 into H2O and O2. The increase in the levels of SOD and CAT in this embodiment indicates that the excessive reactive oxygen species in the cell have activated the scavenging ability of the intracellular antioxidant system. However, the increase in the level of MDA proves that the excessive reactive oxygen species in the cells of Amphidinium robustum have exceeded the scavenging limit of the antioxidant system and have begun to affect the normal structure of the cell membrane.

[0117] 3.3 Effect of DHS on the expression of genes of Amphidinium robustum and the docking of key gene molecules

[0118] In this example, the expression of genes related to carbon absorption and assimilation, nutrient transport and utilization, environmental stress resistance and apoptosis in the robust algae was determined by real-time fluorescence quantitative PCR. Ribulose-1,5-bisphosphate carboxylase / oxygenase II (RuB) is a key enzyme for fixing CO2 in the dark reaction of the photosynthetic system, and is also an oxygenase in the photorespiration metabolic pathway. In this example, the relative expression level of RuB decreased significantly after drug treatment, indicating that DHS can further interfere with the assimilation of organic matter by the algae photosynthetic system by inhibiting the transcription level of the RuB gene in addition to reducing the efficiency of light energy utilization and disrupting the electron transport chain.

[0119] Heat shock protein 90 (HSP90) is a molecular chaperone that plays an important role in helping intracellular proteins fold, maintaining protein spatial structure, and protecting cells from environmental stress. In this example, the transcription level of HSP90 in algae cells treated with DHS was significantly upregulated. Combined with the results of nutrient transporter gene expression in this example, we speculate that the excessive reactive oxygen induced by DHS may have begun to attack macromolecular enzymes in algae cells and cause certain damage to their functional structures. Therefore, cells need to transcribe and express new functional proteins in large quantities. As an important protein for maintaining protein spatial structure, the transcription level of HSP90 is also increased accordingly. The overexpression of HSP90 offsets the stress induced by DHS to a certain extent.

[0120] Arginine / lysine specific cysteine ​​proteases (METs) play an important role in programmed cell death induced by biological (such as pathogen infection, allelopathic effects, etc.) and abiotic (such as nutrient deficiency, osmotic pressure changes, etc.) stresses. Programmed cell death is a gene-controlled and irreversible suicide phenomenon, which aims to promote and maintain the stability of cell genetics and population structure by selectively eliminating certain diseased individuals in the population. Studies have shown that excessive reactive oxygen in algal cells can induce programmed cell death. [6] , the significant upregulation of MET gene expression level in this example indicates that DHS treatment has activated the programmed death of M. robusta, and the growth of the test algal cell population is further affected.

[0121] The strength of the drug's effect on the test subject mainly depends on the ease with which the drug enters the cell and its ability to bind to the target site. The n-octanol-water partition coefficient (lgKow) of the drug characterizes the drug's ability to enter the cell. The larger the lgKow value, the easier it is for the drug to enter the cell. [7] The n-octanol-water partition coefficient of DHS is 5.8. The polar structure of the hydroxyl group in the molecular head makes it easy for the hydrophilic end to insert into the cell membrane phospholipid molecules. Considering the drug's penetrability into various cell membranes and organelle membranes, we used molecular docking to analyze the direct binding of DHS ligands to the key protein receptors of the robust anemone. Generally speaking, receptors with an absolute value of binding energy with the ligand greater than 6.5 can be regarded as potential drug targets. [8] Therefore, in this example, RuB, RHO, and MET may be potential targets for direct action after DHS crosses the membrane and enters the cell. Hydrogen bonds and hydrophobic interactions are the main interactions between DHS and protein receptors. The main function of rhodopsin (RHO) is to generate ATP by capturing light energy to provide energy for other physiological functions such as flagellar movement and material transport. The normal operation of this protein is another important source of energy for photosynthetic algae. Existing studies have shown that hydrogen bonds and hydrophobic bonds in drug structure complexes can weaken the force of target proteins to maintain spatial conformation, causing changes in the spatial microstructure of these proteins, ultimately leading to a decrease in protein catalytic efficiency or even enzyme inactivation. [9] The molecular docking results of this example show that DHS is very likely to cause dysfunction of these enzymes with important physiological functions through direct binding with RuB, RHO, and MET.

[0122] 4. Conclusion

[0123] This example shows that D-dihydrosphingosine is a new and highly efficient natural allelopathic substance, which has a good inhibitory effect on the growth of red tide algae. In terms of half effective control dose, its control effect is one order of magnitude higher than that of common allelopathic substances. The study of the inhibition mechanism shows that D-dihydrosphingosine mainly controls the growth of algae through the following three pathways: 1) reducing the light energy absorption efficiency of algae, interfering with the electron transfer of PSⅠ and PSⅡ reaction centers to affect the light reaction efficiency of algae; 2) inducing excessive reactive oxygen, destroying the antioxidant system of algae, attacking and affecting the normal physiological functions of other intracellular biomacromolecules such as cell membranes; 3) by inhibiting the expression of key genes, reducing the CO2 fixation rate of algae dark reaction, and inducing programmed death of algae.

[0124] Example 8 References:

[0125] [1]NAKAI S,INOUE Y,HOSOMI M.Algal growth inhibition effects and induction modes by plant-producing phenols[J].Water Res,2001,35(7):1855-9.

[0126] [2]MEN YJ,HU HY,LI FM. Effects of the novel allelochemical ethyl 2-methylacetoacetate from the reed (Phragmitis australis Trin) on the growth of several common species of green algae[J].J Appl Phycol, 2007,19(5):521-7.

[0127] [3]HONG Y,HU HY,XIE

[0128] [4] Huang Haomin. Study on the anti-algae activity of natural flavonoids-structure relationship, mechanism of action and development of anti-algae agents[D]; Zhejiang University, 2016.

[0129] [5] Li Chao. Study on the inhibitory effect of flavonoids on the growth and photosynthetic activity of Phaeocystis sphericalensis and quantitative structure-activity relationship model[D]; Zhejiang University, 2019.

[0130] [6]ZHU X,DAO G,TAO Y,et al.A review on control of harmful algalblooms by plant-derived allelochemicals[J].J Hazard Mater,2021,401:123403.

[0131] [7]GEYER H,POLITZKI G,FREITAG D.Prediction of ecotoxicological behavior of chemicals:Relationship between n-octanol / water partitioncoefficient and bioaccumulation of organic chemicals by alga Chlorella[J].Chemosphere,1984,13(2):269-84.

[0132] [8]ZHAO H, GARTENMANN L, DONG J, et al. Discovery of BRD4 bromodomaininhibitors by fragment-based high-throughput docking[J]. Bioorg Med Chem Lett, 2014, 24(11):2493-6.

[0133] [9]LIN W, YAN Y, PING S, et al. Metformin-induced epigenetic toxicity inzebrafish: experimental and molecular dynamics simulation studies [J]. EnvironSci Technol, 2021, 55(3): 1672-81.

[0134] The embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preventing and controlling harmful algae in water bodies using sphingosine substances, characterized in that: Adding sphingosines to water bodies where algae blooms are imminent or have already occurred; The sphingosine substance is selected from 4-hydroxysphingosine, D-sphingosine, D-dihydrosphingosine or N-acetyl-D-sphingosine; The objects of control of harmful algae are selected from the blooms of blue algae and green algae caused by Microcystis aeruginosa and Chlorella vulgaris in freshwater, and the red tides caused by Prosocystis robusta, Phaeocystis sphericalensis, Heterosigma akashiwo, Isochrysis globosum, and Skeletonema costatum in seawater.

2. The method according to claim 1, characterized in that: Use of D-dihydrosphingosine to control red tides dominated by Amphidinium robustum.

3. The method according to claim 1, characterized in that 4-Hydroxysphingosine is used to control red tides dominated by Heterosigma akashiwo, Phaeocystis sphericalensis, Isochrysis globosum or Skeletonema costatum.

4. The method according to claim 1, characterized in that: Use of 4-hydroxysphingosine to control algal blooms dominated by Microcystis aeruginosa or Chlorella vulgaris.

Images