KR101384641B1 - CsNIV capsid containing HPA3NT3 peptide having anti-algae activity against organism induced red tide and the use of the same - Google Patents

Abstract

The present invention relates to the loading of a tide peptide described as HPA3NT3 having a tide activity to CsNIV isolated from red tide, specifically exhibits excellent anti-algae activity, no cytotoxicity, natural degradation and water solubility. By mounting the HPA3NT3 peptide on the CsNIV capsid, the water-soluble HPA3NT3 peptide was easily dissolved in seawater. Therefore, the red tide control agent containing the HPA3NT3 peptide-CsNIV capsid complex as an active ingredient can be used safely and usefully in marine ecosystems. have.

Description

CsNIV capsid containing HPA3NT3 peptide having anti-algae activity against organism induced red tide and the use of the same}

The present invention provides a chaetoceros nucleus encapsulated virus ( Chaetoceros) loaded with HPA3NT3 peptide having a tide activity. salsugineum nuclear inclusion virus (CsNIV) capsids and uses thereof.

When plankton meets an environment suitable for breeding and breeds in large quantities, the phenomenon that the color of seawater turns red, green, or brown due to the various pigments thereof is commonly called red tide. During the rainy season or rainy season in the south coast of Korea, red tide occurs when the sea color turns reddish brown, and sometimes the fish, such as oysters, shellfish, and mussels living in the sea, lose even fish. In addition, when people eat fish and shellfish that eat toxic phytoplankton, various kinds of toxic poisoning occur.

To date, several methods have been studied to prevent red tide or reduce damage caused by red tide. Spraying chemicals (Steidinger, 1983; Ryu et al., 1998) or using an ultrasonic grinder, ozone generator, or centrifugal separator to remove red tide organisms chemically and physically. In addition, a method of adsorbing red tide causative organisms using clay has been developed (Na et al., 1996; Choi et al., 1998). However, the use of this method may have another adverse effect on the ecosystem, and there are many problems to be used in a wide range of red tides.

As another method for solving red tide, research is being conducted to remove red tide causative organisms using biological methods. This is a method of eliminating unwanted organisms in the ecosystem using predator-predator relationships (Ishio et al., 1989; Sakata et al., Fukami et al., 1992; Park et al., 1998). However, this method may change the food chain of the ecosystem and cause other problems. In addition, in other fields, methods for killing red tide by marine bacteria are being actively studied. Recently, attempts have been made to control red tide organisms using protein synthesis inhibitors, cell wall inhibitors, and antibiotics that act on cell membranes. Alternatively, these materials can be modeled to produce artificially low-molecular substances, which can accumulate in the oceans without being degraded, resulting in marine ecosystems and oceans, such as problems caused by pesticides used in agriculture. There is a possibility that it can accumulate in the food produced in the body and even in the human body.

Considering these problems, there are peptides or protein preparations that can be decomposed naturally and can be used as a pharmaceutical preparation that is harmless to the human body or natural ecosystem (Korean Patent Registration 10-1062793). In addition, they can reduce the production cost by mass expression through genetic manipulation, as well as express peptide activity on the surface of bacteria or viruses that specifically attach to red tide organisms. For example, the antimicrobial peptide of Mesto blue ratio (Mastoparan B) having an amphipathic alpha helix purified from bee venom to date red tide organisms in Alexandria Solarium Tama lances (Alexandrium tamarense ) and chattonella catenatum have been shown to have a killing effect. However, the peptides have been reported to show high cytotoxicity at low concentrations.

Chaetoceros has been previously used salsugineum coat protein gene of nuclear inclusion virus (CsNIV) has been studied (Y. Park et al ., Virus Research 142 (2009) 127.133). However, by mounting a specific peptide having a tide activity and water-soluble peptide in the CsNIV capsid isolated from the red tide, it is possible to prevent the specific peptide having the tide activity is easily dissolved in seawater, and maintain the tide activity of the peptide We haven't confirmed anything about it.

Therefore, the inventors of the present invention, HPA3NT3, which is an analog of an increased cation after cleaving the N-terminal part of an irregular (HPA3) peptide having an increased hydrophobic portion, as a parent using HP (2-20) peptide derived from Helicobacter piori When the peptide was mounted on the CsNIV capsid separated from the red tide, it was confirmed that the trough activity of the HPA3NT3 peptide was maintained, thereby confirming that the CsNIV capsid loaded with the HPA3NT3 peptide can be used as an active ingredient of the red tide control agent. Was completed.

An object of the present invention is HPA3NT3 peptide having a tide activity is Chaetoceros nucleus encapsulated virus ( Chetoceros) salsugineum nuclear inclusion virus (CsNIV) capsid, to provide a complex of HPA3NT3 peptide-CsNIV capsid.

Still another object of the present invention is to provide a method for preparing a CsNIV capsid complex on which the HPA3NT3 peptide having the tide activity is mounted.

Still another object of the present invention is to provide a red tide control agent containing the CsNIV capsid complex loaded with the HPA3NT3 peptide having the tide activity.

In order to solve the above problems, the present invention is the HPA3NT3 peptide consisting of the amino acid sequence of SEQ ID NO: 3 is chattoceros salsginium nuclear encapsulated virus ( Chaetoceros salsugineum nuclear inclusion virus (CsNIV) provides a HPA3NT3-CsNIV capsid complex mounted on a capsid.

In addition,

1) digesting the CsNIV capsid with capsomer in a pH 5.5-6.5 buffer;

2) adding an HPA3NT3 peptide; and

3) providing a method for preparing the HPA3NT3-CsNIV capsid complex, comprising the step of forming a CsNIV capsid complex loaded with HPA3NT3 peptide in a buffer at pH 7.5 to 8.5.

In addition, the present invention provides a red tide control agent containing the HPA3NT3-CsNIV capsid complex as an active ingredient.

The present invention relates to a CsNIV capsid loaded with the HPA3NT3 tide peptide, and specifically, is loaded with CsNIV capsid, which is cytotoxic, naturally degradable, and soluble in the tide-induced algae. , It was confirmed that the twitch activity of the HPA3NT3 peptide is maintained. Thus, by mounting the water-soluble HPA3NT3 peptide on the CsNIV capsid, the HPA3NT3 peptide was easily dissolved in seawater. Therefore, the red tide control agent containing the HPA3NT3 peptide-CsNIV capsid complex as an active ingredient is a safe red tide control agent for marine ecosystems. It can be usefully used as an active ingredient of.

1 is a Chaetoceros nuclear encapsulated virus ( Chaetoceros) salsugineum Nuclear inclusion virus (CsNIV) is a diagram showing the method for identifying the HPA3NT3 peptide loaded on:
TEM: transmission electron microscope.
FIG. 2A is an electron microscope view of monomer forms of viruses separated in a capsomer form, and FIG. 2B is an illustration of a monomeric form of the capsomer type virus of FIG. 2A assembled with a CsNIV capsid. It is observed by an electron microscope;
Four colored circles indicate that the size of the assembled capsid is slightly different.
Figure 3a is a diagram observed in the electron microscope as it is mounted on the gold probed when assembled to the capsid in the CsNIV virus capsomer, Figure 3b is a gold probed peptide mounted on the CsNIV virus capsid by the negative staining method Is a drawing;
Black: Gold probed peptide.
4 is a diagram confirming whether the peptide is mounted inside the capsid using a physicochemical fluorescence analysis method.
5 is a diagram showing the effect of the tide activity with the peptide dissociation of the capsid when the peptide mounted in the capsid is treated with Chattonella catenatum , a harmful red tide.

Hereinafter, the present invention will be described in detail.

According to the present invention, the HPA3NT3 peptide having the amino acid sequence of SEQ ID NO: 3 is composed of Chaetoceros nucleus encapsulated virus ( Chetoceros). salsugineum nuclear inclusion virus (CsNIV) provides a HPA3NT3-CsNIV capsid complex with tide activity mounted on a capsid.

The HPA3NT3 peptide may have an amino acid sequence in which the ninth glutamic acid (Glu) and the 13th serine (Ser) are substituted with lysine (Lys) in the HPA3 peptide sequence of SEQ ID NO: 2, but is not limited thereto.

The HPA3 peptide has a sequence of replacing 16th glutamine (Gln) and 18th amino acid aspartic acid (Asp) with tryptophan (Trp) in an HP (2-20) peptide sequence derived from Helicobacter pylori of SEQ ID NO: 1 It may be, but is not limited thereto.

The HPA3NT3 peptides kokeulrodinium poly Cri Koh disk (Cochlodinium polykrikoides, C. polykrikoides), Kobe thiazol Marina (Cobetia marina , C. marina ), Heterosigma akashiwo , H. akashiwo ) and chattonella catenatum ( Chattonella catenatum, C. catenatum ) may be one having at least tide activity against red tide-induced algae selected from the group consisting of, but not limited to.

The CsNIV capsid may be one of the amino acid sequences described in Y. Park et al. (Virus Research 142 (2009) 127.133) included as a reference of the present invention, specifically, may have an amino acid sequence of SEQ ID NO: 4 or 5 .

In a specific embodiment of the present invention, the HP (2-20) peptide derived from Helicobacter pylori, the analog HPA3 peptide substituted with tryptophan at the 16th amino acid glutamine and the 18th amino acid aspartic acid, structurally irregular N- in HPA3 Amphiphilic antibody-binding HPA3NT3 peptides (peptide) having an amino acid sequence substituted with 9th glutamic acid and 13th serine by lysine were prepared in order to increase the cation after cleavage of the terminal region (see Table 1).

In addition,

1) digesting the CsNIV capsid with capsomer in a pH 5.5-6.5 buffer;

2) adding an HPA3NT3 peptide; and

3) providing a method for preparing the HPA3NT3-CsNIV capsid complex, comprising the step of forming a CsNIV capsid complex loaded with HPA3NT3 peptide in a buffer at pH 7.5 to 8.5.

The buffer solution of step 1) may be pH 6.0, but is not limited thereto.

The buffer solution of step 3) may be pH 8.0, but is not limited thereto.

In a specific embodiment of the present invention, in order to mount the tide HPA3NT3 peptide, a chaetoceros salseneum nuclear encapsulated virus ( Chaetoceros) salsugineum nuclear inclusion virus (CsNIV) The contents of Y. Park et al. (Virus Research 142 (2009) 127.133), incorporated by reference herein, were prepared. The amino acid sequence of the main coat protein of CsNIV has been registered in the NCBI protein database as CsNIV ((CsNIV, accession number YP_473363) (SEQ ID NO: 4) and (CsNIV, accession number YP_473362) (SEQ ID NO: 5)).

In order to mount the trough HPA3NT3 peptide to CsNIV, the CsNIV capsid protein was digested into a monomeric or small molecular weight capsomer structure in pH 6 buffer, and then formed into a stable capsid structure in pH 8.0 buffer. Red tide HPA3NT3 peptide was loaded. When the gold (A) gold probe of the HPA3NT3 peptide mounted on the virus by the assembly process, as a result, when confirmed by electron microscopy, it was confirmed that the gold probed HPA3NT3 peptide in the CsNIV capsid (Fig. 1).

The capsomer purified using a Ni-NTA column was assembled without agicide HPA3NT3 peptide, and as a result, the monomer form of the virus separated in capsomer form by electron microscopy was observed (see FIG. 2A). It was confirmed to be assembled into monomeric CsNIV virus capsid (see FIG. 2B). In addition, it was confirmed that the sizes of the assembled CsNIV capsids are different as indicated by the four colored circles in FIG. 2B.

In addition, when the gold-stamped peptides mounted on the CsNIV virus capsids were identified by electron staining using a negative staining method, when the HPA3NT3 peptides were mounted on the CsNIV virus capsomers as capsids, they were examined by electron microscopy. (See FIG. 3A) and, when assembled with capsid in CsNIV virus capsomer, was loaded with gold probed HPA3NT3 peptide and confirmed by negative staining method (see FIG. 3B). As a result, it was confirmed that the gold-probe peptide was present inside the capsid in black. In addition, it was confirmed that the fluorescence probed on the HPA3NT3 peptide present in the CsNIV capsid rapidly dissociated after dissociation of the CsNIV capsid when confirming whether the peptide was loaded inside the capsid by a physicochemical fluorescence analysis method (see FIG. 4).

In addition, the present invention provides a red tide control agent containing the HPA3NT3-CsNIV capsid complex as an active ingredient.

The concentration of the HPA3NT3-CsNIV capsid complex may be showing the tide activity when the concentration of 0.01 μM to 100 μM, and specifically, when the concentration of the HPA3NT3-CsNIV capsid complex is 0.25 μM to 1.00 μM. However, it is not limited thereto.

The red tide control agent may have anti-red tide activity against one or more red tide-induced algae selected from the group consisting of coclodinium polycricoidis, covetia marina, flagella and chatonella catenium.

In a specific embodiment of the present invention, in order to confirm that the HPA3NT3 peptide loaded in the CsNIV capsid has a tide effect, the CsNIV capsid loaded with the HPA3NT3 peptide was treated in a harmful red tide Chattonella catenatum , and CsNIV. It was confirmed that the HPA3NT3 peptide inside the capsid destroyed harmful red tide with the dissociation of the CsNIV capsid (see FIG. 5).

Thus, by mounting the HPA3NT3 peptide, which exhibits excellent anti-algae activity, is free of cytotoxicity, is naturally degraded, and is soluble in the CsNIV capsid, the water-soluble HPA3NT3 peptide can be easily dissolved in seawater, thereby preventing the HPA3NT3 peptide- Red tide control agents containing CsNIV capsid complex as an active ingredient can be used safely and usefully in marine ecosystems.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. These Examples and Experimental Examples are only for illustrating the present invention, and the scope of the present invention is not limited by these Examples and Experimental Examples.

< Example  1> Trace Of peptide  Synthesis and separation purification

In order to synthesize amphiphilic tide peptides, HPA2 peptide derived from Helicobacter pylori, an analog HPA3 peptide substituted with tryptophan with glutamine (16th amino acid) and aspartic acid (18th amino acid), HPA3 In order to increase the cation after cutting the structurally irregular N-terminal site, HPA3NT3 peptide was prepared having an amino acid sequence substituted with 9th glutamic acid and 13th serine with lysine (Table 1).

Amino acid sequence of the peptide used in the test Peptide Name order Molecular Weight  HP (2-20) AKKVFKRLEKLFSKIQNDK-NH ₂ (SEQ ID NO: 1) 2320.0  HPA3 AKKVFKRLEKLFSKIWNWK- NH ₂ (SEQ ID NO: 2) 2449.2  HPA3NT3 FKRLKKLFKKIWNWK- NH ₂ (SEQ ID NO: 3) 2062.6

The peptide was synthesized using the liquid phase solidification method of Merrifield using 9-fluorenylmethoxycarbonyl (Fmoc) as an amino group protective container. The carboxyl terminus of the peptide of the present invention is -NH ₂ Peptide in form was used as a starting material Link Amide MBHA-Resin (Rink Amide MBHA-Resin). The extension of the peptide chain by coupling of Fmoc-amino acids is based on the N-hydroxybenzo triazole-dicyclo-hexycarbodiimide (HOBt-DCC). ). After coupling the Fmoc-amino acid of each peptide amino terminal, the Fmoc group was removed with 20% piperidine / dimethyl formamide (hereinafter abbreviated as 'DMF') solution, and DMP and dichoromethane, DCM) several times and dried with nitrogen gas. Trifluoroacetic acid (TFA) on the dried resin: phenol: thioanisole: water (H ₂ O): triisopropylsilane = 85: 5: 5: 2.5 : 2.5 (vol./vol.) Solution was added and reacted for 4 hours to remove the protecting group, the peptide was separated from the resin, and then reacted with cold diethyl ether for 30 minutes, followed by centrifugation of the peptide. Precipitated and again precipitated peptide precipitated with diethyl ether and dried in air. The crude peptide obtained through the above process was dissolved in ammonium bicarbonate (pH 8), and acetonitrile was used as a gradient to reverse phase-HPLC column ( Delta Pak, C ₁₈ 300 mm 3, 19.0 mm x 30 cm, Waters). Purified synthetic peptides were measured with an amino acid analyzer (Hitachi 8500 A) using the Edman degradation method, and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The molecular weight of the peptide was measured.

As a result, all the peptides synthesized in the present invention showed a purity of 95% or more, and it was confirmed that the peptides having the correct amino acid sequence were synthesized by matching the molecular weights calculated from the amino acid sequences.

< Example  2> Chattoceros Salsginum  Nuclear encapsulated virus ( Chaetoceros salsugineum nuclear inclusion virus , CsNIV A) Preparation of the coat protein

Based on the contents of Y. Park et al. (Virus Research 142 (2009) 127.133) included as a reference of the present invention, CsNIV coat proteins were prepared as follows.

Chattoceros salsginium Ch42 was inoculated with purified CsNIV and incubated, after which CsNIV pellets were collected. Primers were designed to amplify the gene encoding the CsNIV coat protein, and polymerase chain reaction (PCR) was performed using the DNA of CsNIV as a template. A vector containing a gene encoding the CsNIV coat protein was prepared, and the CsNIV coat protein was purified after expression in E. coli . The purified CsNIV coat proteins were analyzed by mass spectrometry, and the BLASTX program (http://www.ncbi.nlm.nih.gov/BLAST) was used to compare partial amino acid sequences and known protein databases. Was used to confirm the amino acid sequence. The amino acid sequence of the main coat protein of CsNIV has been registered in the NCBI protein database as CsNIV ((CsNIV, accession number YP_473363) (SEQ ID NO: 4) and (CsNIV, accession number YP_473362) (SEQ ID NO: 5)).

< Example  3> Trace HPA3NT3 Of peptide CsNIV Mount of furnace

CsNIV capsid protein is composed of monomeric or small molecular weight capsomer structures in pH 6 buffer (20 mM sodium phosphate, 150 mM NaCl, 0.5% (v / v) Triton X-100, pH 6.0). And a stable capsid structure in pH 8.0 buffer (20 mM Tris-Hcl, 150 mM NaCl, 1 mM CaCl ₂ , 0.5% (v / v) Triton X-100, pH 8.0). When it is decomposed | disassembled into the small molecular weight capsomer structure as mentioned above, and forms a stable capsid structure, it is a wake bath HPA3NT3. Peptides were loaded.

< Experimental Example  1> CsNIV  Mounted in a virus HPA3NT3 Of peptide  gold( gold ) Probe Confirmation

Gold particles (AuNP-citrate, 20 nm) were purchased from Sigma-Aldrich and 6.99 × 1011 for the experiment. The particles were used in units of particles / ml (520 nm = 0.91). This was reacted with HPA3NT3, the algae peptide prepared in <Example 1>, and the condition was dissolved in 0.05 mM HPA3NT3 peptide in injectable-grade water and mixed with 1 nM of gold particles, followed by CsNIV under an electron microscope. The capsid was used for the purpose of confirming whether the HPA3NT3 peptide was loaded. At this time, a molar ratio of approximately AuNP / peptide 1: 5000 was optimal.

As a result, when confirmed by electron microscopy, it was confirmed that the gold-probed HPA3NT3 peptide was loaded in the CsNIV capsid (FIG. 1).

< Experimental Example  2> CsNIV  virus Cap Somer  In mode CsNIV virus Capsidro  Assembly

The capsomer purified using a Ni-NTA column was assembled without the algae HPA3NT3 peptide.

As a result, it was possible to observe the monomer form of the virus separated in the capsomer form by electron microscopy (Fig. 2a), it was confirmed that the assembly of monomeric CsNIV virus capsid (Fig. 2b). In addition, it was confirmed that the sizes of the assembled CsNIV capsids are different as indicated by the four colored circles in FIG. 2B.

< Experimental Example  3> Negative Staining ( negative staining Method using an electron microscope CsNIV  virus Capsy Mounted on Gold fall Probed Of peptide  Confirm

When assembled to capsid in CsNIV virus capsomer, either HPA3NT3 peptide or HPA3NT3 peptide with gold probe was loaded and observed by electron microscopy to confirm this.

As a result, the HPA3NT3 peptide was mounted on capsid in CsNIV virus capsomer and confirmed by electron microscopy (FIG. 3a). Also, when the capsid was assembled on CsNIV virus capsomer, gold probed HPA3NT3 peptide was loaded and negative. Confirmation by staining method (FIG. 3B). As a result, it was confirmed that the gold-probe peptide was present inside the capsid in black.

< Experimental Example  4> By physicochemical fluorescence analysis Of peptide Capsid  Check whether it is mounted inside

Physicochemical fluorescence analysis was used to determine whether HPA3NT3 peptide was present inside the CsNIV capsid. In other words, if the Rhodamin probed HPA3NT3 peptide was mounted inside the CsNIV capsid, the fluorescence spectroscopy did not appear at the beginning of the experiment, but if the CsNIV capsid was broken by artificial treatment, the fluorescence would be rapidly increased. Rhodamine probed HPA3NT3 peptide was mounted inside the CsNIV capsid and the degree of fluorescence before and after artificial treatment was measured.

As a result, it was confirmed that the fluorescence probed in the HPA3NT3 peptide existing inside the CsNIV capsid appeared rapidly after dissociation of the CsNIV capsid (FIG. 4).

< Experimental Example  5> Capsid  Was inside Of peptide  Hazardous Red Tide Experiment

To see if it has an anti-bloom effect of the peptide with HPA3NT3 in CsNIV capsid, was treated with a HPA3NT3 CsNIV capsid peptide with the toxic blooms of Chateau Nella category natyum (Chattonella catenatum). At this time, the harmful red tide Chattonella catenatum ( Chattonella catenatum ) was prepared in a 24-well tissue culture plate (24-well tissue culture plate) adjusted to 2 ~ 4 × 10 ⁴ cells / mL. The HPA3NT3 peptide in the experimental CsNIV capsid dissolved in f / 2 medium (Sigma-Aldrich) was adjusted to a concentration of 0.25 μM and observed under a microscope (Olympus, Tokyo, Japan) one hour after the peptide treatment.

As a result, when the HPA3NT3 peptide loaded in the CsNIV capsid was treated with the harmful red tide, Chattonella catenium, it was confirmed that the HPA3NT3 peptide inside the CsNIV capsid destroyed the harmful red tide with the dissociation of the CsNIV capsid (FIG. 5).

Attach an electronic file to a sequence list

Claims (10)

HPA3NT3-CsNIV capsid complex, wherein the HPA3NT3 peptide, consisting of the amino acid sequence of SEQ ID NO: 3, is mounted in a Chaetoceros salsugineum nuclear inclusion virus (CsNIV) capsid.
The method of claim 1, wherein the HPA3NT3 peptide is an amino acid sequence of the 1 to 4 amino acids deleted from the HPA3 peptide sequence of SEQ ID NO: 2, the ninth glutamic acid (Glu) and the 13th serine (Ser) by lysine (Lys) HPA3NT3-CsNIV capsid complex having a wake-up activity, characterized in that consisting of.
3. The HPA3 peptide according to claim 2, wherein the HPA3 peptide comprises 16th glutamine (Gln) and 18th amino acid aspartic acid (Asp) as tryptophan (Trp) in the HP (2-20) peptide sequence derived from Helicobacter pylori of SEQ ID NO: 1. HPA3NT3-CsNIV capsid complex having a tide activity, characterized in that consisting of a substituted sequence.
The method of claim 1, wherein the HPA3NT3 peptide is Cochlodinium ( Cochlodinium) polykrikoides , C. polykrikoides ), Cobetia marina ( C. marina ), Heterosigma akashiwo , H. akashiwo ) and Chattonella catenatum ( Chattonella catenatum, C. catenatum ) HPA3NT3-CsNIV capsid having a tide activity, characterized in that there is a tide activity against at least one red tide-induced algae selected from the group consisting of Complex.
The HPA3NT3-CsNIV capsid complex according to claim 1, wherein the CsNIV capsid is composed of the amino acid sequence of SEQ ID NO: 4 or 5.
1) digesting the CsNIV capsid with capsomer in a buffer at pH 5.5-6.5;
2) adding an HPA3NT3 peptide; and
3) A method for preparing the HPA3NT3-CsNIV capsid complex according to claim 1, comprising the step of forming a CsNIV capsid complex loaded with the HPA3NT3 peptide in a buffer at pH 7.5 to 8.5.
The method of claim 6, wherein the buffer solution of step 1) is pH 6.0, the method for producing the HPA3NT3-CsNIV capsid complex according to claim 1.
The method of claim 6, wherein the buffer solution of step 3) is pH 8.0, wherein the HPA3NT3-CsNIV capsid complex according to claim 1.
Red tide control agent containing the HPA3NT3-CsNIV capsid complex according to claim 1 as an active ingredient.
10. The method according to claim 9, wherein the red tide control agent has a tide activity against at least one red tide-induced algae selected from the group consisting of coclodinium polycricoidis, covetia marina, flagella and chatonella catenium. Red tide control.

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