A method for preparing a transmission electron microscopy sample of the epidermis of Phenacoccus solenopsis Tinsley

Abstract

The present invention provides a method for preparing a transmission electron microscopy (TEM) sample of the cuticle of Phenacoccus solenopsis. Specifically, the present invention provides a method for preparing a TEM sample of an insect cuticle, the method comprising the following steps: sampling, rinsing, dehydration, infiltration, drying, sectioning, and then staining the sectioned sample with a lead citrate solution to obtain a TEM sample of the cuticle of Phenacoccus solenopsis.

Description

Technical Field

[0001] The present invention relates to the field of agricultural science. Specifically, the present invention provides a method for preparing a transmission electron microscopy sample of the epidermis of Phenacoccus solenopsis Tinsley. Background Art

[0002] Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is a worldwide invasive pest. It has strong adaptability to abiotic stresses in the environment, can damage more than 100 kinds of crops and garden plants, and is difficult to control, which is related to its characteristics such as rich wax secretion on its body surface, wide host range, and strong reproductive ability. Currently, the main method for controlling Phenacoccus solenopsis Tinsley is to spray chemical pesticides, that is, chemical control. As the epidermis is the barrier for insects to directly contact the outside world, it will directly affect the control effect.

[0003] In China, there is less research on the epidermal structure of cotton scale insects. Most of the research on the integument of scale insects focuses on Ericerus pela (Hemiptera: Coccidae). Because its female insects adapt to overwintering by thickening the epidermis and have extremely strong wax secretion ability, it has become a research hotspot. For Pseudococcidae insects, researchers mostly focus on the epidermal wax secretion of Pseudococcidae insects, and often use scanning electron microscopy to observe glandular structures on the epidermis, etc. There is no relevant research on using transmission electron microscopy technology to study the epidermal structure of Phenacoccus solenopsis Tinsley.

[0004] The insect epidermis consists of three main composite layers, namely the outer envelope, the middle epicuticle, and the inner procuticle. The envelope is composed of lipids and proteins, and can form protein-quinone complexes through covalent bonding, and its structure is very stable. There is no obvious thickening of the cuticle in the epidermis of Phenacoccus solenopsis Tinsley, and in addition, its body size is small, so the conventional sample preparation method cannot clearly distinguish the envelope and the epicuticle.

[0005] In view of this, the present invention proposes a transmission electron microscopy sample preparation method that can effectively distinguish the envelope structure of the epidermis of Phenacoccus solenopsis Tinsley through tests, and can be applied and promoted to the preparation of transmission electron microscopy samples for the study of the outer epidermal structure of Pseudococcidae. Summary of the Invention

[0006] The purpose of the present invention is to make improvements based on the existing technology, and provide a method for preparing a transmission electron microscopy sample of the epidermis of Phenacoccus solenopsis Tinsley and its application. The method for preparing a transmission electron microscopy sample of the epidermis of Phenacoccus solenopsis Tinsley of the present invention can directly, objectively, and accurately reflect and distinguish the envelope in the epidermal structure of Phenacoccus solenopsis Tinsley.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is: providing a method for preparing a transmission electron microscopy sample of the epidermis of Phenacoccus solenopsis Tinsley, including the following steps:

[0008] Sampling: Dissect the cuticle of a live insect sample and remove the contents to obtain a cuticle sample of the insect body;

[0009] Rinsing: Rinse the cuticle sample of the insect body one or more times;

[0010] Dehydration: Dehydrate the rinsed sample to obtain a dehydrated cuticle sample of the insect body;

[0011] Infiltration: After treating the sample with a mixture of Spurr embedding medium and acetone, infiltrate the sample with the embedding medium;

[0012] Drying: Place the treated sample in pure Spurr embedding medium, pose it, and heat it to dry;

[0013] Sectioning: Section the sample;

[0014] Staining: Stain the sectioned sample with lead citrate solution to obtain a transmission electron microscopy sample of the cuticle of Phenacoccus solenopsis Tinsley.

[0015] In a preferred embodiment, the concentration of the lead citrate solution is 0.05 - 0.2 M.

[0016] As a preferred embodiment of the preparation method of the present invention, in the sampling step, the dissection is a live dissection. When dissecting, the ventral surface of the insect body is not completely cut open. While removing the tissue contents, the morphology of the cuticle is not damaged, which can prevent the cuticle from shrinking and deforming in subsequent steps.

[0017] As a preferred embodiment of the preparation method of the present invention, after the dissection is completed, place the sample on filter paper to dry the sample, and then immediately put it into a 1.5 ml centrifuge tube containing 2.5% glutaraldehyde fixative.

[0018] As a preferred embodiment of the preparation method of the present invention, after the sample collection is completed, centrifuge briefly to precipitate the sample to the bottom of the tube, and make the sample fully contact the fixative. Carefully aspirate the upper fixative and add new fixative.

[0019] In a preferred embodiment, the rinsing step includes: fixing the cuticle sample of the insect body in glutaraldehyde fixative, then rinsing with PBS buffer; then immersing the sample in osmium tetroxide and rinsing with buffer again.

[0020] As a preferred embodiment of the preparation method of the present invention, when rinsing, invert the centrifuge tube up and down to rinse it thoroughly. Before replacing the PBS solution, let the sample stand still to sink to the bottom of the tube. When aspirating the old solution, keep the pipette tip close to the tube wall and carefully aspirate the solution to prevent the sample from being aspirated away.

[0021] In a preferred embodiment, the dehydration treatment includes: dehydrating the sample in an ethanol solution with a gradient concentration, and then treating it with ethanol and acetone respectively to obtain a dehydrated cuticle sample of the insect.

[0022] The wax dissolves in organic substances, and the dehydration step directly affects the cuticular wax of insects. Therefore, whether the fixation is sufficient, the dehydration concentration and time are very important for the quality of the sections. The present invention strictly controls the dehydration concentration and time. In a preferred embodiment, the ethanol concentration gradient successively includes:

[0023] Treating with 25 - 35% (v / v) aqueous ethanol solution for 15 min;

[0024] Treating with 45 - 55% (v / v) aqueous ethanol solution for 15 min;

[0025] Treating with 65 - 75% (v / v) aqueous ethanol solution for 15 min;

[0026] Treating with 75 - 85% (v / v) aqueous ethanol solution for 15 min;

[0027] Treating with 85 - 95% (v / v) aqueous ethanol solution for 15 min;

[0028] Treating with 90 - 100% (v / v) aqueous ethanol solution for 15 min.

[0029] This is a preferred embodiment of the preparation method of the present invention. The alcohol concentrations used in S4 are successively 30% (v / v), 50% (v / v), 70% (v / v), 80% (v / v), 90% (v / v), 95% (v / v), and the rinsing method is the same as the above operation.

[0030] In a preferred embodiment, the infiltration treatment includes: treating the sample of S4 with a 0.8 - 1.2:0.8 - 1.2 (v / v) mixture and a 2 - 4:0.8 - 1.2 (v / v) mixture of Spurr embedding medium and acetone respectively, and then infiltrating the sample with Spurr embedding medium.

[0031] In a preferred embodiment, the drying treatment includes: posing the sample in pure Spurr embedding medium, and then heating it to dry at 60 - 80 °C.

[0032] In a preferred embodiment, the sectioning includes: obtaining sections with a thickness of 60 - 80 nm on a microtome and collecting them on a carbon - coated copper grid.

[0033] In a preferred embodiment, the staining method is as follows: completely immerse the sections in a lead citrate solution for 5 - 20 min, then wash the sections with double-distilled water and air-dry.

[0034] In a preferred embodiment, the method comprises the steps of:

[0035] S1. Sampling: Pick a live insect sample, quickly dissect the epidermis of the insect under a dissecting microscope, remove the contents, and obtain a sample.

[0036] S2. Fixation and rinsing: Immediately immerse the sample from S1 in a 2.5% glutaraldehyde fixative, store at 4°C for 8 - 10 h, then rinse 3 times with 0.1 M PBS buffer, 15 min each time.

[0037] S3. Re-fixation and rinsing: Completely immerse the sample from S2 in 1% osmium tetroxide, treat at room temperature for 1.5 h, then rinse 3 times with 0.1 M PBS buffer, 15 min each time.

[0038] S4. Dehydration: Dehydrate the sample from S3 in ethanol solutions of gradient concentrations for 15 min, then treat with 100% ethanol and 100% acetone for 20 min each.

[0039] S5. Infiltration: At room temperature, treat the sample from S4 with a 1:1 mixture and a 3:1 mixture of Spurr embedding medium and acetone for 1 h and 3 h respectively, then infiltrate the sample with pure embedding medium overnight for 9 h.

[0040] S6. Drying: Place the sample from S5 in pure Spurr embedding medium for posing, heat at 70°C for more than 9 h.

[0041] S7. Orientation and sectioning: Section the sample from S6 that has been oriented with an ultramicrotome, and collect the sections on a grid.

[0042] S8. Staining, observation, and photography: Stain the sample from S7 with a lead citrate solution (0.1 M) for 10 min, observe under a high-resolution transmission electron microscope, and take pictures.

[0043] As a preferred embodiment of the preparation method of the present invention, S7 specifically is: Obtain 70-nm sections with a LEICA EM UC7 ultramicrotome, and collect the sections on a 100-mesh copper carrier grid with a carbon film.

[0044] As a preferred embodiment of the preparation method of the present invention, the staining method in S8 is as follows: Drop a drop of 0.1 M lead citrate solution on a silica gel staining pad, carefully place the copper mesh on the droplet with forceps, completely immerse the sections in the staining solution, stain for 10 min, then wash the sections with double-distilled water and air-dry on filter paper.

[0045] As a preferred embodiment of the preparation method of the present invention, the electron microscopy observation in S8 is specifically as follows: Observation is carried out under JEM-2100plus (JEOL, Japan), the acceleration voltage is 200 kv, and the image is captured using a gatan Rio9 type CMOS camera and the picture results are saved.

[0046] As a preferred embodiment of the preparation method of the present invention, the observation object is applicable to Phenacoccus solenopsis Tinsley.

[0047] Compared with the prior art, the advantages of the present invention include:

[0048] 1. The observation of the epidermal structure mostly exists in insects with a relatively thick cuticle. There is less research on the epidermal structure of insects represented by Phenacoccus solenopsis Tinsley, which secretes wax as a protective layer. Since the degree of keratinization of its epidermal structure is relatively low, it is not easy to distinguish the outer envelope from other structures. The present invention hopes to improve the conventional transmission electron microscopy sample preparation and staining method, which is conducive to observing and distinguishing the outer envelope from other epidermal layers. The application of this technology to Phenacoccus solenopsis Tinsley is the first time, and it has important reference significance for the research and application of other mealybug insects.

[0049] 2. The present invention provides technical support for distinguishing the outer envelope in the transmission electron microscopy sample preparation of the epidermis of Phenacoccus solenopsis Tinsley. When sampling, there is no need to pretreat the insect body, which reduces the damage to its epidermal wax. For the sample acquisition method of the present invention, after dissecting the insect body and removing the contents, the fixing solution can penetrate into the interior of the insect body for epidermal fixation, enhancing the epidermal penetration effect and making it easy to distinguish the orientation of the insect body in the subsequent epidermal treatment, which is convenient for observing and positioning special positions of the epidermis. The present invention proposes an operation method after obtaining the sample: After brief centrifugation, the supernatant is aspirated to replace the fixing solution, so that the sample can precipitate to the bottom of the tube and fully contact the fixing solution to make it fixed sufficiently. The staining method of the present invention is improved on the conventional uranyl acetate-lead citrate double staining method, and only lead citrate solution is used for staining, which makes it easy to distinguish the outer envelope of the insect epidermis from other structures.

[0050] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0051] Figure 1Transmission electron microscopy photographs of the cuticles of female adults of Phenacoccus solenopsis treated with different staining methods. A: After sectioning, the dye is 0.1 M lead citrate solution; B: After sectioning, the dye is 50% ethanol saturated solution of uranyl acetate; C: After sectioning, the dye is 0.1 M lead citrate solution and 50% ethanol saturated solution of uranyl acetate; D: No staining treatment after sectioning. Outer envelope (env); Epicuticle (epi); Procuticle (pro). Detailed implementation manners

[0052] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0053] Unless otherwise specified, all kit materials used in the following embodiments are commercially available.

[0054] General method: Preparation of 100 ml of 0.1 M PBS buffer

[0055] Solution a: Weigh 31.61 g of Na2HPO4·H2O and add double-distilled water to 1000 ml; Solution b: Weigh 27.6 g of NaH2PO4·H2O and add double-distilled water to 1000 ml; Take 61 ml of solution a and 39 ml of solution b and mix them into a 0.2 M PBS buffer stock solution, and then dilute it 1-fold with double-distilled water to obtain 0.1 M PBS buffer (pH 7.0).

[0056] Example 1

[0057] I. Method:

[0058] Sampling: Use a small brush to pick several female adults of Phenacoccus solenopsis and put them into a petri dish containing 0.1 M PBS buffer. Quickly dissect the cuticle of the insect body under a dissecting microscope, remove the contents, wash it 3 times in clean PBS buffer, and then blot the liquid dry on filter paper to obtain a sample;

[0059] Fixation and rinsing: Immediately immerse the above sample in a centrifuge tube containing 1 ml of 2.5% (wt) glutaraldehyde fixative, centrifuge at low speed (3000 rpm) in a centrifuge for 1 min to make the sample sink to the bottom of the tube. Carefully remove the fixative with a pipette against the wall of the tube (to prevent sucking the sample), then add fresh fixative and store it at 4°C for 8 h - 10 h. When rinsing, carefully suck the fixative with a dropper against the wall of the tube, add 0.1 M PBS buffer, let it stand for 15 min, and frequently invert the centrifuge tube up and down during this period to make the sample fully contact with the buffer. Repeat this rinsing step 3 times;

[0060] Fixation and rinsing: After completely sucking dry the buffer solution of the above sample, 1% (wt) osmium tetroxide was dropped in, and the sample was fully immersed and treated at room temperature for 1.5 hours. Then it was rinsed 3 times with 0.1M PBS buffer solution, 15 minutes each time, and the rinsing method was the same as above. Adding osmium tetroxide and the first rinsing were carried out in a fume hood.

[0061] Dehydration: The above sample was dehydrated with ethanol solutions with gradient concentrations of 30% (v / v), 50% (v / v), 70% (v / v), 80% (v / v), 90% (v / v), and 95% (v / v), 15 minutes for each concentration gradient, and then treated with 100% ethanol and 100% acetone for 20 minutes respectively. The operation method was the same as rinsing (that is, after adding the corresponding treatment solution, let it stand for 15 minutes, and during this period, often invert the centrifuge tube up and down to make the sample fully contact with the treatment solution, repeat 3 times). The dehydration steps strictly controlled the time.

[0062] Infiltration: At room temperature, the above sample was treated with a 1:1 mixture and a 3:1 mixture of Spurr embedding agent and acetone for 1 hour and 3 hours respectively, and then infiltrated with pure embedding agent overnight for 9 hours.

[0063] Drying: The above sample was posed in pure Spurr embedding agent, with the head position of the insect body epidermis facing upward and the insect body placed vertically in the embedding agent, and heated at 70°C for more than 9 hours.

[0064] Positioning and sectioning: The above sample with good positioning was sliced into 70nm sections with a LEICA EM UC7 ultramicrotome, and the sections were collected on a 100-mesh copper grid with a carbon film.

[0065] Staining, observation, and photographing: A drop of 0.1M lead citrate solution was dropped on a silica gel staining pad, and the copper grid was carefully placed on the liquid drop with pointed forceps, and the sections were completely immersed in the staining solution for 10 minutes. Then the sections were washed with double-distilled water and dried on filter paper. Observation was carried out under a JEM-2100plus (JEOL, Japan), the accelerating voltage was 200kv, and image acquisition was performed using a gatanRio9 type CMOS camera.

[0066] II. Results

[0067] The transmission electron microscopy sample of the adult epidermis of Phenacoccus solenopsis prepared by this method can clearly show the cross-section of the overall structure of the epidermis. Figure 1 Figure A shows the cross-section of the outer envelope, epicuticle layer, and procuticle layer of the epidermis of female adult Phenacoccus solenopsis. The results show that the outer envelope of the cotton scale insect epidermis is the outermost and thinner layer of structure, and the innermost procuticle layer has abundant chitin fibers arranged in parallel, and the epicuticle is in between for transition.

[0068] Study on Staining Method of Comparative Example 1 (I)

[0069] I. Method

[0070] Compared with the Example, the only difference is that in S8, the section staining only uses the 50% ethanol saturated solution of uranyl acetate for staining treatment.

[0071] II. Results

[0072] As Figure 1 shown in Fig. B, since uranyl acetate can bind to most molecules in the cell, although the outer envelope, epicuticle and procuticle can be roughly distinguished, the boundary between each layer is blurred and it is not easy to observe with the naked eye. When the magnification of the electron microscope is relatively high, the sample is blurred and it is difficult to distinguish the outer envelope and the epicuticle.

[0073] Study on Staining Method of Comparative Example 2 (II)

[0074] I. Method

[0075] Compared with the Example, the only difference is that in S8, the section staining uses 0.1M lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 10 min of staining treatment respectively.

[0076] II. Results

[0077] As Figure 1 shown in Fig. C, the conventional double heavy metal staining can enhance the contrast between various structures in the sample, but in the epidermal structure of Phenacoccus solenopsis, the overall color of the sample is darker. Although the epicuticle layer and the procuticle can be clearly distinguished, it is difficult to distinguish the outer envelope and the epicuticle layer. When the magnification of the electron microscope is relatively high, the sample is blurred and difficult to distinguish.

[0078] Study on Staining Method of Comparative Example 3 (III)

[0079] I. Method

[0080] Compared with the Example, the only difference is that in S8, the section is no longer stained additionally.

[0081] II. Results

[0082] As Figure 1 shown in Fig. D, since no heavy metal staining is carried out, although the outer envelope, epicuticle layer and procuticle can be roughly distinguished, the boundary between each layer is blurred, the overall contrast of the sample is poor, and it is difficult to clearly observe the outer envelope.

[0083] All the documents mentioned in the present invention are cited in this application as references, just as if each document is cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for preparing a transmission electron microscopy sample of insect cuticle, characterized in that, The method includes the following steps: Sampling: Dissect the cuticle of a live insect sample and remove the contents to obtain a cuticle sample of the insect; Rinsing: Rinse the cuticle sample of the insect one or more times; Dehydration: Perform dehydration treatment on the rinsed sample to obtain a dehydrated cuticle sample of the insect; Infiltration: After treating the sample with a mixture of Spurr embedding medium and acetone, infiltrate the sample with the embedding medium; Drying: Place the treated sample in pure Spurr embedding medium, pose it, and heat it to dryness; Sectioning: Section the sample; Staining: Stain the sectioned sample with a lead citrate solution to obtain a transmission electron microscopy sample of the cuticle of Phenacoccus solenopsis; The concentration of the lead citrate solution is 0.05 - 0.2 M; the insect is Phenacoccus solenopsis.

2. The method according to claim 1, characterized in that, The rinsing step includes: Fix the cuticle sample of the insect in a glutaraldehyde fixative, then rinse it with a PBS buffer solution; then immerse the sample in osmium tetroxide and rinse it with the buffer solution.

3. The method according to claim 1, wherein The dehydration treatment includes: Perform dehydration treatment on the sample in an ethanol solution with a gradient concentration, and then treat it with ethanol and acetone respectively to obtain a dehydrated cuticle sample of the insect.

4. The method according to claim 3, characterized in that, The ethanol concentration gradient successively includes: Treat with a 25 - 35% (v / v) aqueous ethanol solution for 10 - 20 min; Treat with a 45 - 55% (v / v) aqueous ethanol solution for 10 - 20 min; Treat with a 65 - 75% (v / v) aqueous ethanol solution for 10 - 20 min; Treat with a 75 - 85% (v / v) aqueous ethanol solution for 10 - 20 min; Treat with an 85 - 95% (v / v) aqueous ethanol solution for 10 - 20 min; Treat with a 90 - 100% (v / v) aqueous ethanol solution for 10 - 20 min.

5. The method according to claim 1, wherein The infiltration treatment includes: After treating the sample in step S4 with a 0.8 - 1.2:0.8 - 1.2 (v / v) mixture and a 2 - 4:0.8 - 1.2 (v / v) mixture of Spurr embedding medium and acetone respectively, infiltrate the sample with Spurr embedding medium.

6. The method according to claim 1, wherein The drying treatment includes: Place the sample in pure Spurr embedding medium, pose it, and then heat it to dryness at 60 - 80 °C.

7. The method according to claim 1, characterized in that The sectioning includes: Obtain sections with a thickness of 60 - 80 nm on a microtome and collect them on a carbon - coated copper grid.

8. The method according to claim 1, characterized in that, The staining method is: Immerse the sections completely in the lead citrate solution for staining for 5 - 20 min, then wash the sections with double - distilled water and air - dry them.

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