A test method for adhesion of jellyfish nematocysts and its application

Abstract

The present invention discloses a method for testing the adhesion of jellyfish nematocysts and its application. The method comprises the following steps: isolating nematocysts: subjecting a jellyfish suspension to density gradient centrifugation using Percoll centrifuge to obtain nematocysts; preparing a coating: applying different coating materials to the center of a culture dish to form a smooth coating; adhering nematocysts: dropping the separated nematocysts onto the center of the coating and covering it with a coverslip for a predetermined period of time; staining: removing the coverslip, adding acridine orange as a dye, covering it with a coverslip to allow the dye to spread, and staining for a predetermined period of time; and image processing: photographing the sample using an inverted fluorescence microscope and processing the image using image processing software to obtain the fluorescently stained area. The method uses acridine orange for staining, resulting in specific staining, clear images, and easy identification of nematocysts. The results can be digitized. This method is rapid, simple to use, and highly operational, making it easy to screen for jellyfish sting protective coatings.

Description

Technical Field

[0001] The present invention relates to the field of marine biotechnology, and in particular to a method for testing the adhesion of jellyfish nematocysts and an application thereof. Background Art

[0002] In recent years, jellyfish outbreaks have become common worldwide, and jellyfish stings have become a significant risk factor for maritime activities such as coastal tourism, marine fishing, and naval training. Clinical symptoms of jellyfish stings include tingling and burning sensations, followed by erythematous rashes. In severe cases, stings can lead to chest tightness, shortness of breath, and even shock and death. Jellyfish are cnidarians, and their tentacles contain numerous different types of nematocysts. Nematocysts are a type of organelle unique to cnidarians, primarily spherical, fusiform, and ellipsoidal in shape. Nematocysts consist of an outer wall, internal inverted tubules, and a toxin stored within. Receptors are located on the surface of the nematocyst wall. Upon mechanical or chemical stimulation, the small lid at the top of the nematocyst opens. Driven by the significant osmotic pressure difference between the inside and outside, the inverted tubules are ejected at extremely high speeds, rapidly penetrating the stratum corneum and epidermis of the human skin, injecting toxins into the body and causing a series of skin lesions and other symptoms. Upon contact between tentacles and skin, numerous nematocysts remain on the skin surface, most of which remain unreleased.

[0003] Therefore, preventing the adhesion of jellyfish sting cysts is the core of jellyfish sting protection research. Testing the adhesion of jellyfish sting cysts is key to jellyfish sting protection research and a prerequisite for screening protective coatings. To date, no testing method has been reported, so the development of a testing method is necessary. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a test method and application of the adhesion of jellyfish nematocysts, so as to achieve the purpose of specific staining, digitized results, simple operation and fast process.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] A method for testing the adhesion of jellyfish nematocysts comprises the following steps:

[0007] Step 1: Isolation of nematocysts: Density gradient centrifugation of the jellyfish suspension using Percoll centrifuge to obtain nematocysts;

[0008] Step 2: Coating preparation: Apply different coating materials to the center of the culture dish to form a smooth coating;

[0009] Step 3, nematocyst adhesion: drop the separated nematocysts onto the center of the coating and cover with a cover glass for a certain period of time;

[0010] Step 4: Staining: After removing the coverslip, add acridine orange stain and cover with a coverslip to allow the stain to spread. Stain for a set period of time.

[0011] Step 5: Image processing: Take pictures using an inverted fluorescence microscope and process them using image processing software to obtain the fluorescent staining area.

[0012] In the above scheme, in step 3, the adhesion time is 15-90s.

[0013] In the above scheme, in step 3, a weight is placed on the coverslip to provide pressure during adhesion.

[0014] In the above scheme, in step 4, the staining time is 5-10 minutes.

[0015] In the above scheme, in step three, 2.5 μL of nematocysts is added dropwise, and in step four, 5 μL of the dye acridine orange is added dropwise.

[0016] In the above scheme, in step 5, the image processing software is ImageJ, and the fluorescent staining area is obtained through the Analyze Particles function of the software.

[0017] In the above scheme, in step 5, the adhesion center area and multiple fields of view around it are selected for photographing, and the total staining area is obtained by adding them up, and the staining area is used as an indicator of the adhesion of the nematocysts.

[0018] A jellyfish sting cyst adhesion test method is used to evaluate the adhesion behavior of different coatings to jellyfish sting cysts, helping to screen jellyfish sting protection coatings.

[0019] Through the above technical solution, the present invention provides a method for testing the adhesion of jellyfish nematocysts and its application, which has the following beneficial effects:

[0020] 1. The method of the present invention uses acridine orange as a dye for staining, which is specific, produces clear images, and the nematocysts are easily distinguishable, and the results can be digitized.

[0021] 2. The method of the present invention has a fast process, simple equipment, and strong operability, and is convenient for screening jellyfish sting protective coatings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0023] Figure 1 A schematic diagram of the adhesion and staining process in a method for testing the adhesion of jellyfish nematocysts disclosed in an embodiment of the present invention;

[0024] Figure 2 This is a photo processed by the image processing software ImageJ in Example 1 of the present invention;

[0025] Figure 3 These are dyeing photos of Example 1 and Examples 5-9 of the present invention, wherein (a)-(f) correspond to Example 1 and Examples 5-9, respectively. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present invention.

[0027] Example 1

[0028] Step 1: Isolation of nematocysts: Density gradient centrifugation of the jellyfish suspension using Percoll centrifuge to obtain nematocysts;

[0029] Step 2: Prepare gelatin coating:

[0030] Weigh 2g of gelatin solid particles into a beaker and add 18mL of distilled water. Allow to swell for 20 minutes, then stir with a magnetic stirrer and heat to 60°C to dissolve. Once dissolved, add 1mL of 1% glutaraldehyde for cross-linking. Heat to 60°C and stir for 40 minutes to obtain a gelatin solution.

[0031] 1 mL of the prepared gelatin solution was added to a 30 mm culture dish. The dish was tilted to evenly distribute the gelatin on the bottom of the dish. The dish was naturally cured for 10 h to form a gelatin skin model.

[0032] Step 3, nematocyst adhesion: 2.5 μL of the separated nematocysts was dropped into the center of the gelatin coating and slowly covered with a cover glass for 15 seconds; Figure 1 As shown in (a)-(c);

[0033] Step 4: Staining: After removing the coverslip with tweezers, add 5 μL of acridine orange (AO) and cover with a coverslip to allow the stain to spread. Stain for 5 minutes. Figure 1 As shown in (d)-(f);

[0034] Step 5, image processing: Use an Olympus inverted fluorescence microscope to select the adhesion center area and 9 surrounding fields to take pictures, and use the image processing software ImageJ to process the pictures. Adjust the contrast, exposure value, brightness and other parameters to make the nematocyst boundary clearly visible, such as Figure 2 As shown, the fluorescent staining area was obtained by the Analyze Particles function of the software.

[0035] Example 2

[0036] The difference between this embodiment and embodiment 1 is that step 3 is adhered for 60 seconds and step 4 is dyed for 8 minutes.

[0037] Example 3

[0038] The difference between this embodiment and embodiment 1 is that step 3 is adhered for 90 seconds and step 4 is dyed for 10 minutes.

[0039] Example 4

[0040] The difference between this embodiment and embodiment 1 is that in step 3, a 50 g weight is placed on the cover glass and adhered for 15 seconds. The remaining operations are the same as those in embodiment 1.

[0041] Comparative Example 1

[0042] The difference between this comparative example and Example 1 is that the adhesion step is 10 seconds and the dyeing step is 4 minutes.

[0043] Comparative Example 2

[0044] The difference between this comparative example and Example 1 is that step 3 adhesion is performed for 100 seconds, and step 4 dyeing is performed for 12 minutes.

[0045] For each example and comparative example, five parallel experiments were performed (i.e., steps 3 and 4 were repeated five times). Nine fields of view of the adhesion center area were selected for each parallel experiment. The total staining area (μm) was obtained by summing the staining areas in the fields of view. 2 ), with the staining area as the data representing the amount of nematocyst adhesion. The staining area data results of the above examples and comparative examples are shown in Table 1.

[0046] Table 1 Staining area data (μm 2 )

[0047] Parallel 1 Parallel 2 Parallel 3 Parallel 4 Parallel 5 Mean ± SD Example 1 214031 178134 189107 156375 212738 190077±24324 Example 2 884912 776782 876219 874097 987006 879803±74460 Example 3 1090008 1099149 1051880 1083080 1005769 1065977±38060 Example 4 394240 466280 320523 414311 399041 398879±52303 Comparative Example 1 37967 51499 30341 36129 77424 46672±18859 Comparative Example 2 462431 350970 360848 418917 370249 392683±46930

[0048] As can be seen from Table 1, the adhesion time of Examples 1-3 was within 15-90 seconds, and the staining time was within 5-10 minutes. As the adhesion time and staining time increased, the fluorescent staining area reached a relatively stable state. The adhesion time and staining time of Comparative Example 1 were lower than the adhesion time and staining time specified in the present invention, and the fluorescent staining area was too low. After the adhesion time and staining time of Comparative Example 2 exceeded the adhesion time and staining time specified in the present invention, the fluorescent staining area did not change significantly. In Example 4, the adhesion time could be appropriately shortened after applying pressure on the cover glass.

[0049] The adhesion test method of the present invention can be used to evaluate the adhesion behavior of different coatings to jellyfish sting cysts, thereby helping to screen jellyfish sting protective coatings. Specific examples are as follows:

[0050] Example 5

[0051] The difference between this embodiment and embodiment 1 is that, in step 2, the coating is prepared by applying a small amount of vaseline in the center of a 30 mm culture dish and placing it in a 60° C. oven for 30 seconds to form a flat surface. The remaining operations are the same as in embodiment 1.

[0052] Example 6

[0053] The difference between this embodiment and embodiment 1 is that, in step 2, the coating is prepared by applying a small amount of lanolin in the center of a 30 mm culture dish and placing it in a 60° C. oven for 30 seconds to form a flat surface. The remaining operations are the same as those in embodiment 1.

[0054] Example 7

[0055] The difference between this embodiment and embodiment 1 is that in step 2, the coating is prepared by pouring a small amount of PDMS in the center of a 30 mm culture dish, tilting the culture dish so that it is evenly spread on the bottom of the dish, and curing it in a 60° C. oven for 2 hours. The remaining operations are the same as in embodiment 1.

[0056] Example 8

[0057] The difference between this embodiment and embodiment 1 is that in step 2, the coating is prepared by applying a small amount of 10% PVA prepared with distilled water to the center of a 30 mm culture dish and allowing the film to form naturally at room temperature for 1 hour to form a PVA coating. The remaining operations are the same as those in embodiment 1.

[0058] Example 9

[0059] The difference between this embodiment and embodiment 1 is that, in step 2, the coating is prepared by dropping 200 μL of PU in the center of a 30 mm culture dish and spreading it evenly, and the film is formed naturally at room temperature for 1 hour. The rest of the operations are the same as those in embodiment 1.

[0060] In the above examples, 5 parallels were performed for each example (i.e., the operations of steps 3 and 4 were repeated 5 times). Nine fields of view of the adhesion center area were selected for each parallel, and the stained areas in the fields of view were summed to obtain the total stained area (μm 2 ), the dyeing area is used as the data to represent the amount of nematocyst adhesion. The dyeing area data results of the above embodiment are shown in Table 2, and the dyeing photos are shown in Table 2. Figure 3 As shown in (a)-(g).

[0061] Table 2 Staining area data (μm 2 )

[0062] Parallel 1 Parallel 2 Parallel 3 Parallel 4 Parallel 5 Mean ± SD Example 1 214031 178134 189107 156375 212738 190077±24324 Example 5 77656 79597 86557 75590 86329 81146±5040 Example 6 132432 98540 128875 107259 140854 121592±17877 Example 7 29622 24881 21284 35559 22054 26680±5942 Example 8 128628 124483 139984 103424 125400 124384±13245 Example 9 30889 30711 30622 27106 29641 29794±1579

[0063] As can be seen from Table 2, under the same conditions, the adhesion of nematocysts to gelatin coating is the greatest, the adhesion to vaseline, lanolin and PVA is decreased, and the adhesion to PDMS and PU is the least. Therefore, PDMS and PU can be preliminarily screened as protective coatings for jellyfish stings.

[0064] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for testing the adhesion of jellyfish nematocysts, characterized in that: The steps include: Step 1: Isolation of nematocysts: Density gradient centrifugation of the jellyfish suspension using Percoll centrifuge to obtain nematocysts; Step 2: Coating preparation: Apply different coating materials to the center of the culture dish to form a smooth coating; Step 3, nematocyst adhesion: drop the separated nematocysts onto the center of the coating and cover with a cover glass for a certain period of time; Step 4: Staining: After removing the coverslip, add acridine orange stain and cover with a coverslip to allow the stain to spread. Stain for a set period of time. Step 5, image processing: take pictures using an inverted fluorescence microscope and process them using image processing software to obtain the fluorescent staining area; In step 3, a weight is placed on the coverslip to provide pressure during adhesion; In step 5, the adhesion center area and multiple fields of view around it are selected for photographing, and the total staining area is obtained by adding them up, and the staining area is used as an indicator of the adhesion of the nematocysts.

2. The method for testing the adhesion of jellyfish nematocysts according to claim 1, wherein: In step 3, the adhesion time is 15-90s.

3. The method for testing the adhesion of jellyfish nematocysts according to claim 1, wherein: In step 4, the staining time is 5-10 minutes.

4. The method for testing the adhesion of jellyfish nematocysts according to claim 1, wherein: In step three, 2.5 μL of nematocysts was added dropwise, and in step four, 5 μL of acridine orange dye was added dropwise.

5. The method for testing the adhesion of jellyfish nematocysts according to claim 1, wherein: In step 5, the image processing software is ImageJ, and the fluorescent staining area is obtained through the Analyze Particles function of the software.

6. An application of the method for testing the adhesion of jellyfish nematocysts according to any one of claims 1 to 5, characterized in that: Used to evaluate the adhesion behavior of different coatings to jellyfish sting cysts, helping to screen protective coatings against jellyfish stings.

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