Application of homosalate in jellyfish bloom prevention and control

Abstract

The present invention belongs to the technical field of jellyfish prevention and control, and discloses the application of homosalate in the prevention and control of jellyfish outbreaks. Homosalate is safe and non-toxic at low concentrations, has a low cost, is simple and easy to operate, and has a quick effect. Homosalate has great potential in the application of jellyfish outbreak prevention and control. The above method of the present invention is applicable to coastal fishing grounds, tourist waters, and waters near nuclear power plants where jellyfish outbreaks occur, and can be used to prevent and control jellyfish outbreaks. It is recommended to sprinkle an appropriate concentration of homosalate in the waters in advance.

Description

Technical Field

[0001] The present invention relates to the technical field of jellyfish prevention and control, and more specifically, to the application of Homosalate in the prevention and control of jellyfish blooms. Background Art

[0002] Jellyfish are one of the most harmful marine organisms to humans. The losses caused by jellyfish blooms include multiple aspects, and the three main ones are as follows: First, due to jellyfish blooms, the number of stung people has increased significantly in coastal areas. On the one hand, jellyfish stings bring a heavy medical burden and the treatment effect is not obvious; on the other hand, due to jellyfish blooms, the output of the fishing industry has decreased sharply, bringing inestimable losses to the fishing industry and the tourism industry.

[0003] Homosalate, also known as homosalate (English: Homosalate, chemical formula: C 16 H 22 O3), also known as trimethylcyclohexyl salicylate, trimethyl salicylate, homosalate, is a colorless transparent liquid chemical substance that is insoluble in water and can absorb ultraviolet rays in the UVB 295-315 wavelength band. It is mainly used as an ultraviolet UVB sunscreen in industry. Existing technical reports show that Homosalate also significantly inhibits the invasion of HTR8 / SVneo cells. In addition, it regulates the phosphoinositide 3-kinase (PI3K) / AKT and mitogen-activated protein kinase (MAPK) signaling pathways.

[0004] Using Homosalate inhibition as an effective prevention and control means for jellyfish blooms has not been reported yet. Summary of the Invention

[0005] In order to overcome the above problems existing in the prior art, the application of Homosalate as a metamorphosis inhibitor of Aurelia aurita is provided first.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] The present invention first provides the application of Homosalate as a metamorphosis inhibitor of Aurelia aurita.

[0008] The present invention also provides the application of Homosalate in the preparation of functional products for preventing and controlling Aurelia aurita blooms.

[0009] Through experimental verification, in the artificially prepared seawater with chemical induction and the artificial seawater inducer added with 1 μM Homosalate, the group added with the Homosalate inhibitor can significantly reduce the transverse fission rate of polyps at the same time, and at the ephyra stage, significantly reduce the number of ephyrae, thereby reducing the number of jellyfish.

[0010] Therefore, preferably, in the above application, Homosalate has the following effects:

[0011] (1) Reduce the early transverse fission rate of the metamorphosis of Aurelia aurita, and / or,

[0012] (2) Reduce the number of ephyrae of Aurelia aurita, and / or,

[0013] (3) Slow down the transverse fission and proliferation rate of the polyps of Aurelia aurita.

[0014] More preferably, in the above application, the concentration of Homosalate is 100 nM to 1 μM.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The inhibitor Homosalate can inhibit the metamorphosis process of the polyps of Aurelia aurita in a chemical inducer. Through experiments, it is verified that soaking the polyps of Aurelia aurita with 100 nM of Homosalate inhibitor and 1 μM of Homosalate inhibitor can both inhibit metamorphosis. The results show that starting from the second day, the number of transverse fissions in the experimental group is less than that in the control group, and the number of ephyrae appearing is also reduced compared with the control group. This provides a theoretical basis for the prevention and control of jellyfish outbreaks.

[0017] In addition, Homosalate is safe and non-toxic at low concentrations, has low cost, is simple and easy to operate, and has a quick effect. Homosalate has great potential in the application of jellyfish outbreak prevention and control. The above method of the present invention is applicable to coastal fishing grounds, tourist waters, and waters near nuclear power plants where jellyfish outbreaks occur, and can be used to prevent and control jellyfish outbreaks. It is recommended to sprinkle an appropriate concentration of Homosalate in the waters in advance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shows the effects of 1 μM and 100 nM of Homosalate inhibitors and the control group on the metamorphosis of Aurelia aurita at different stages;

[0019] Figure 2 Shows the production amounts of Aurelia aurita of 1 μM and 100 nM of Homosalate inhibitors and the control group at corresponding stages on different days. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0022] 1. Experimental Materials

[0023] Preparation of artificial brine: Dissolve sea salt in tap water, stir with a glass rod, use an optical salinity meter to measure the salinity at 2.8‰-3.2‰, and let it stand to remove debris.

[0024] Preparation of chemical inducer: Weigh 0.0403 g of 5-methoxy-2-methylindole (Solebol), place it in a 5 L container bottle, add 5 L of artificial seawater, and shake until completely dissolved, thus preparing 50 μM 5-methoxy-2-methylindole chemical inducer.

[0025] 20℃ constant temperature box; others: culture dish, corrugated plate, water tank, 24-well plate.

[0026] 2. Experimental Methods

[0027] Experiment 1: Extraction of total protein from polyp and jellyfish stages

[0028] 1) Sample acquisition: Use a disposable pipette to scrape along the corrugated plate from top to bottom, use the pipette to suck the polyps from the corrugated plate and put them into a beaker filled with seawater, rinse the polyps with seawater several times to wash away impurities, and finally rinse them three times with 1× PBS.

[0029] 2) Homogenization: Collect the cleaned moon jellyfish polyps, add an equal volume of 1×PBS solution, and homogenize using a homogenizer at 4°C for 20 seconds × 3 times, with an interval of 15 seconds each time.

[0030] 3) Stirring: After obtaining the homogenate of the moon jellyfish polyp, transfer it to a beaker and add pre-cooled (4°C) 1×PBS in a volume ratio of about 1:5 between the polyp and 1×PBS. Place the sample in a 4°C refrigerator and stir it with a magnetic stirrer for 72 hours to allow it to fully autolyze.

[0031] 4) Filtration: The autolyzed solution was collected and filtered twice using a 200-mesh sieve, and placed in a 500 ml beaker to remove the undissolved portion and impurities.

[0032] 5) Centrifugation: Collect the filtrate and centrifuge it. Transfer the filtrate to a 50 mL centrifuge tube and centrifuge it at 4°C, 1000×g for 10 min. Collect the supernatant.

[0033] 6) Dialysis: After centrifugation, cut a section of dialysis tubing and boil it in boiling water for 10 min. After boiling, use forceps to pick up the dialysis tubing from the boiling water, clamp one end with a fixing clip, pour the collected supernatant into the dialysis tubing, then clamp the other end with a fixing clip, invert it up and down to check if it is clamped tightly, place it in a beaker, pour 1×PBS into the beaker until the dialysis tubing is submerged, put it into a magnetic stirrer, and dialyze for 1 day. Aliquot it into 15 ml centrifuge tubes and store the venom samples in a -80°C refrigerator.

[0034] 7) Re-centrifugation: Collect the dialysis solution and centrifuge it. Transfer the filtrate to a 50 mL centrifuge tube and centrifuge it at 4°C and 1000×g for 10 min. Collect the supernatant in a 50 ml centrifuge tube. Aliquot it into 15 ml centrifuge tubes to obtain the total protein of hydra. Store the venom samples in a -80°C refrigerator.

[0035] Experiment 2: Toxicity evaluation of mice

[0036] Select ICR mice, male mice weighing 18 - 22 g and 7 weeks old. Inject the extracted total protein with a concentration of 0.3036 mg / ml via the tail vein, and the injection dose is 10 ml / kg. The control group is also injected with the same dose of 1×PBS.

[0037] The injection volume of the experimental group and the control group into the footpads is proportionally reduced according to the body weight of the mice. Record the survival of the mice at 0 h, 0.5 h, 1 h, 2 h, 4 h, 12 h, 24 h, 36 h, and 48 h after injection respectively.

[0038] Experiment 3: Screening of inhibitor concentration

[0039] According to the results of the preliminary experiment, design 2 groups of experiments, namely Homosalate inhibitors with concentrations of 1 μM and 100 nM. On a 12-well plate, there are 3 wells in each group, and 10 pre-selected hydras are placed in each well.

[0040] Preparation of Homosalate inhibitor: Take 1 mg of Homosalate (TaoShu) inhibitor, add 381.1702 μL of DMSO to prepare a 10 mM stock solution ①. Then take 2 μL of stock solution ① and add it to 19998 μL of a 50 nM 5-methoxy-2-methylindole solution prepared with artificial seawater to prepare a 20 ml 1 μM Homosalate inhibitor solution ②.

[0041] Take 1 ml of ② and add it to 9 ml of a 50 nM 5-methoxy-2-methylindole solution prepared with artificial seawater to obtain a 100 nM Homosalate inhibitor solution ③.

[0042] In the wells of the 12-well plate where hydras were added, the three wells in the leftmost column served as the control group, with 3 ml of 50 nM 5-methoxy-2-methylindole solution added to each well. The three wells in the middle column served as the 100 nM homosalate inhibitor group, with 3 ml of ② added to each well. The three wells in the rightmost column served as the 1 μM homosalate inhibitor group, with 3 ml of ③ added to each well. Observe the metamorphosis at the same time every day, and record the time and number of early, late strobilae, and ephyrae produced until all ephyrae autolyze. Then plot a graph to compare the effects of the two concentrations of inhibitors on metamorphosis and select the better one.

[0043] Experiment 4: Homosalate Inhibitor Experiment

[0044] Select hydras that grow well in the water tank with fully extended tentacles, starve them for one day in advance, and then carefully place them in the 12-well plate, with 10 hydras placed in each well. Divide them into a control group and an experimental group. The control group is added with a chemical inducer without an inhibitor, and the experimental group is added with a chemical inducer containing 1 μM homosalate inhibitor. Each group has n = 30, divided into 3 wells, and 10 hydras are added to each well.

[0045] On the second day, take out the samples prepared the previous day, place them under an optical microscope to observe the tentacle extension state of the hydras, and ensure that the growth states of the experimental hydras are all in good condition. Use a disposable pipette to wash the artificial seawater in the 12-well plate clean, quickly add the corresponding inducer and 1 μM inhibitor, make good marks, and place them in an incubator at 20 °C. Record the strobilation of the hydras at the same time every day. For example, record at 10 o'clock on the first day, and then record at 10 o'clock every day until all the ephyrae are completely released.

[0046] In the 12-well plates of the inhibitor group and the control group, 3 ml of artificial seawater, inhibitor, and inducer are added to each well, and the prepared solutions are all from the same batch.

[0047] The standard for early strobilation is from the appearance of the strobilation disc to five discs. The late strobila has a brownish body trunk disc and the discs have not fallen off. The ephyra is marked when the discs have completely fallen off and the individual can freely expand and contract in the solution in a shape similar to a disc.

[0048] 3. Result Analysis

[0049] Through Experiments 1 and 2, it was found that after injecting mice with the total protein of hydras, the mice died successively from 12 minutes to 30 minutes, indicating that the toxicity of the hydra stage of Aurelia aurita is relatively strong.

[0050] Through Experiment 3, we found that there were significant differences in the metamorphosis of hydras treated with different concentrations of Homosalate inhibitor and those not treated with different concentrations of the inhibitor. The effect of the 1 μM Homosalate inhibitor was more obvious than that of the 100 nM Homosalate inhibitor. On the first day of observation, the developmental state of all hydras remained at the polyp stage. On the second day of observation, the number of early strobilae in the control group was 15, in the 100 nM Homosalate group was 13, and in the 1 μM Homosalate inhibitor group was 12. On the third day of observation, the number of early strobilae in the 100 nM Homosalate inhibitor group and the control group was 21, and in the 1 μM Homosalate inhibitor group was 24. On the fourth day of observation, all hydras in the control group had metamorphosed into the early strobila stage. The number of early strobilae in the 100 nM Homosalate group was 27, and in the 1 μM Homosalate inhibitor group was 28. On the fifth day of observation, all three groups were in the early strobila stage. On the sixth day of observation, 2 hydras in the control group developed into late strobilae, 6 hydras in the 100 nM Homosalate group developed into late strobilae, and all hydras in the 1 μM Homosalate inhibitor group remained in the early strobila stage. On the seventh day of observation, 15 hydras in the control group developed into late strobilae, 27 hydras in the 100 nM Homosalate group developed into late strobilae, and only 10 hydras in the 1 μM Homosalate inhibitor group developed into late strobilae. From the ninth to the eleventh day of observation, the number of ephyrae produced in the 1 μM Homosalate inhibitor group was 7, 56, and 70 respectively, while that in the control group was 13, 77, and 85 respectively. Moreover, it was observed that the peak of ephyra production in the 1 μM Homosalate inhibitor group was significantly delayed, occurring on the twelfth and thirteenth days, with the number of ephyrae produced being 84 and 77 respectively, while the peak of ephyra production in the control group was on the tenth day. This indicates that the 1 μM Homosalate inhibitor has an obvious inhibitory effect on metamorphosis.

[0051] Through Experiment 4, we found that there was no difference between the experimental group and the control group on the first day, and both were in the polyp stage. Starting from the second day, 12 early transverse fissions appeared in the experimental group, while there were 15 early transverse fissions in the control group. The transverse fission rate of the control group was 50%, and that of the experimental group was 43.33%. On the third day, 24 polyps in the experimental group underwent transverse fission, with a transverse fission rate of 80%, while only 21 polyps in the control group underwent transverse fission, with a transverse fission rate of 70%. On the fourth day, 28 transverse fissions occurred in the experimental group and 30 in the control group. On the fifth day, all polyps in both the experimental group and the control group underwent transverse fission into early transverse fission bodies. On the sixth day, 30 in the experimental group remained as early transverse fission bodies, while 2 in the control group had already undergone transverse fission into late transverse fission bodies. On the seventh day, 10 in the experimental group developed into late transverse fission bodies, while 13 in the control group were late transverse fission bodies. On the eighth day, 25 in the experimental group were late transverse fission bodies and 4 were medusae, while there were 2 medusae in the control group. On the ninth day, there were 28 late transverse fission bodies and 13 medusae in the control group, 3 early transverse fission bodies, 26 late ones, and 7 medusae in the experimental group. On the tenth day, there were 20 late ones and 77 medusae in the control group, 3 early ones, 17 late ones, and 56 medusae in the experimental group. On the eleventh day, there were 12 late ones and 85 medusae in the control group, 12 late ones and 70 medusae in the experimental group. On the twelfth day, there were 9 late ones and 72 medusae in the control group, 8 late ones and 77 medusae in the experimental group. On the thirteenth day, there were 51 medusae in the control group, 2 late ones and 29 medusae in the experimental group. It shows that the effect of the inhibitor is obvious.

Claims

1. Use of homosalate as an inhibitor of the metamorphosis of Aurelia aurita.

2. Use of homosalate in the preparation of a functional product for controlling the outbreak of Aurelia aurita.

3. The application according to claim 1 or 2, characterized in that, Homosalate has the following effects: (1) Reducing the early transverse fission rate of the metamorphosis of Aurelia aurita, and / or, (2) Reducing the number of ephyrae of Aurelia aurita, and / or, (3) Slowing down the transverse fission and proliferation rate of the polyps of Aurelia aurita.

4. The application according to claim 3, characterized in that, The concentration of homosalate is 100 nM to 1 μM.

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