A method for collecting and purifying the mouthpart secretions of a predatory natural enemy, oratoria

Abstract

The application discloses a method for collecting and purifying mouthpart secretion of a predatory natural enemy Orius minor, and belongs to the technical field of biology. The method comprises the following steps: constructing a double-layer membrane sandwich artificial feeding device, encapsulating a nutrient solution in a sandwich space between a first membrane layer and a second membrane layer, and using the feeding drive of the Orius minor to make the Orius minor actively pierce the membrane layer and feed the nutrient solution, so that the mouthpart secretion is released into the nutrient solution in the natural feeding process; after the nutrient solution containing the secretion is collected, ultrafiltration centrifugation technology is used for concentration and purification, and a high-purity mouthpart secretion protein sample is obtained. The natural feeding state of the Orius minor is simulated, the collected secretion can more truly reflect the functional protein released by the Orius minor in the predation process, meanwhile, the integrated design of collection and purification effectively removes small molecular impurities in the sample, and the sample can be directly used for high-precision analysis such as proteomics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for collecting and purifying the mouthpart secretions of the predatory insect *Stachys macrantha*. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Ming Xiaohua bug ( Orius nagaii Thrips (Pterygota spp.) is a small, predatory insect with piercing-sucking mouthparts that exhibits significant pest control effects in biological control. This insect not only has outstanding predatory abilities against small pests such as thrips, spider mites, aphids, and whiteflies, but can also effectively feed on the eggs and larvae of various lepidopteran pests, making it of significant value in integrated pest management (IPM).

[0004] Studies have shown that the feeding behavior of piercing-sucking insects is closely related to their oral secretions (OS). These secretions contain abundant functional proteins that play crucial roles in lubricating the stylet, digesting food externally, and regulating host defense responses. As an important predatory natural enemy, elucidating the protein composition of the oral secretions of the small-flowered bug not only helps to reveal its predation mechanism but also has significant application value for the development of novel biopesticides and the scientific control of pests. Currently, there have been some technological attempts to collect the oral secretions of piercing-sucking insects. For example, Lü Limin et al. studied a device for collecting oral secretions from piercing-sucking insects. This device utilizes the insect's phototaxis to induce it to pierce a single-layer transparent membrane, thereby obtaining the secretions from the collection liquid on the membrane surface. However, this device has the following shortcomings: (1) Phototaxis-induced membrane-piercing behavior is not the insect's natural feeding behavior, and the collected secretions may not represent the proteins released during actual feeding; (2) The collection liquid is located on the membrane surface, which is easily contaminated by the environment and cannot simulate the liquid environment during insect feeding; (3) This device only involves the collection step and does not provide a subsequent purification scheme. The collected samples have a high impurity content, which is difficult to meet the requirements of high-precision analysis such as proteomics. In addition, since the small size of the *Mallotus maculatus* (approximately 2-3 mm) results in very little mouthpart secretion, there is currently no efficient method for collecting and purifying mouthpart secretions specifically for this species. At present, research on the protein composition and function of mouthpart secretions of *Mallotus maculatus* remains blank.

[0005] Therefore, establishing an efficient and stable method for collecting and purifying the mouthpart secretions of the *Solanum macranthum* is crucial. This method must ensure that the *Solanum macranthum* releases mouthpart secretions during natural feeding, completely capture the secreted proteins, and possess the ability to purify even trace samples, thus providing high-quality samples for subsequent research. The establishment of this technology will advance in-depth research on the interaction mechanisms between predatory natural enemies and pests, and provide important technical support for discovering new pest control targets, possessing significant theoretical and applied value. Summary of the Invention

[0006] The technical solution adopted in this invention is as follows: A first aspect of the present invention provides a method for collecting and purifying the mouthpart secretions of the predatory natural enemy, the small-flowered bug, comprising the following steps: (1) Constructing an artificial feeding device: The device includes a container, a first membrane layer covering the opening of the container, and a second membrane layer disposed above the first membrane layer; a closed interlayer space is formed between the first membrane layer and the second membrane layer, and the interlayer space contains a nutrient solution that can be sucked by the stinger of the *Symplocos macrantha*; the test *Symplocos macrantha* is placed in the container; (2) The small flower bug pierces the first membrane layer to feed on the nutrient solution in the interlayer space. During this process, the small flower bug releases mouthpart secretions into the nutrient solution. The nutrient solution is collected to obtain a sample containing the mouthpart secretions. The nutrient solution is a sugar solution. (3) Place the sample collected in step (2) into an ultrafiltration centrifuge tube for centrifugation and concentration, discard the filtrate, then invert the inner tube of the ultrafiltration centrifuge tube for a second centrifugation, collect the concentrate, and obtain the purified oral secretion sample.

[0007] In one embodiment of the present invention, step (1), constructing the artificial feeding device specifically includes: (a) Preparation of test insects: Collect several healthy small flower bugs (nymphs or adults) and transfer them to sterile petri dishes for later use; (b) Pretreatment of sealing film: Take Parafilm film and stretch it evenly in both directions until it can completely cover the opening of the culture dish. Two intact stretch films are required for each set of devices. (c) Primary sealing: The first layer of extended film is smoothly covered over the opening of the petri dish, ensuring that the edges are well sealed and the film surface is wrinkle-free, so as to prevent the insects from escaping. (d) Feeding system construction: Nutrient solution is evenly dripped onto the surface of the primary sealing film in the form of droplets, and then covered with a second layer of extended film to form a sandwich structure, so that the small flower bug can obtain nutrients by piercing and sucking the nutrient solution in the Parafilm membrane sandwich. Finally, the petri dish is covered and inverted.

[0008] In step (1), the nutrient solution is a sugar solution.

[0009] Preferably, the sugar solution is a sucrose solution with a mass fraction of 1% to 5%. More preferably, it is 2.5%.

[0010] In step (2), the tested pygmy bugs are starved before being placed in the container.

[0011] Preferably, the starvation treatment lasts for 12 to 48 hours.

[0012] In step (3), the *Ming Xiaohua Bug* is cultured for 12 to 48 hours at 20–30°C, relative humidity of 50%–90%, in complete darkness or low light conditions.

[0013] In step (3), the molecular weight cutoff of the ultrafiltration centrifuge tube is 1 to 10 kDa.

[0014] In step (3), the conditions for centrifugal concentration are: temperature 4-10℃, rotation speed 8,000-12,000 rpm, and time 10-30 minutes; the conditions for secondary centrifugation are: rotation speed 3,000-8,000 rpm and time 1-5 minutes.

[0015] In a second aspect of the invention, a sample of mouthpart secretions from the predatory natural enemy *Stachys maculatus* is provided, which is collected and purified using the method described in the first aspect.

[0016] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) Simulate natural feeding conditions and collect real functional secretions. This invention constructs a double-membrane sandwich structure to encapsulate nutrient solution between two membranes, enabling the *Solanum micranthum* bug to actively pierce and suck food driven by hunger, releasing mouthpart secretions during natural feeding. The collected samples more accurately reflect the protein secretion characteristics of the *Solanum micranthum* when hunting prey, providing more biologically significant samples for subsequent proteomics analysis.

[0017] (2) The collection and purification are integrated to achieve high-purity preparation of trace samples. Existing technologies only involve the collection step and do not provide a purification solution, resulting in high impurity content in the collected samples, which is difficult to meet the requirements of high-precision analysis such as proteomics. This invention organically integrates artificial feeding collection with ultrafiltration centrifugation purification, forming an integrated collection-purification technology. By using ultrafiltration centrifuge tubes with a molecular weight cutoff of 1–10 kDa combined with a two-step centrifugation method (concentration followed by recovery), small molecule impurities such as sucrose are effectively removed, yielding high-purity oral secretion protein samples that can be directly used for proteomics analysis such as liquid chromatography-mass spectrometry (LC-MS / MS).

[0018] (3) Starvation treatment synergistically induces protein collection efficiency. The present invention further preferably involves starving the *Symplocos macrantha* for 12–48 hours before collection. Experimental data show that after starvation treatment, the protein concentration significantly increased from 0.4556–0.6863 mg / mL in the untreated group to 0.78–0.80 mg / mL. Figure 3 D). Starvation treatment and the double-membrane sandwich feeding system produced a synergistic effect, effectively promoting the mouthpart secretion activity of the small flower bug and increasing the protein yield per unit sample.

[0019] (4) It is easy to operate, low in cost, and has good repeatability. The materials used in this invention are all commonly used laboratory consumables (petition dishes, Parafilm membranes, sucrose solution, ultrafiltration centrifuge tubes), requiring no special equipment, with simple operation steps and low cost. Under optimized culture conditions (26±1℃, 70±5% relative humidity, complete darkness, 24 h), detectable amounts of oral secretion proteins can be stably obtained with good reproducibility, making it suitable for large-scale sample preparation.

[0020] (5) Promote research on the interaction mechanism between natural enemies and pests This invention establishes for the first time a highly efficient method for collecting and purifying the mouthpart secretions of the tiny predatory bug *Spodoptera litura*, filling a technological gap in this field. The obtained high-purity mouthpart secretion protein samples can be used to analyze the predation mechanism of *Spodoptera litura*, identify key functional proteins, and discover novel pest control targets, possessing significant theoretical and applied value for the development of biological control products and the scientific control of agricultural pests. Attached Figure Description

[0021] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 This is a schematic diagram of the artificial feeding device for the small flower bug and its feeding effect. Wherein: A is a schematic diagram of the artificial feeding device; B is a photograph of the small flower bug's piercing and sucking feeding behavior; C is a fluorescence detection result image of the small flower bug after feeding on a GFP-containing sucrose solution (BF: bright field, GFP: green fluorescence).

[0023] Figure 2 This is a diagram showing the process for collecting and purifying the secretions from the mouthparts of the small-flowered bug, and the results of protein concentration determination.

[0024] Wherein: A is a schematic diagram of the process of collecting oral secretions and purifying them by ultrafiltration and centrifugation; B is a comparison diagram of the colorimetric reaction of BCA protein concentration (from left to right: blank control, OS-1, OS-2, OS-3); C is a standard curve of BCA protein concentration determination (horizontal axis: protein concentration mg / mL, vertical axis: OD562 absorbance). D is a bar chart of the calculated protein concentration results for the samples (OS-1, OS-2, OS-3).

[0025] Figure 3 This is a graph showing the effect of starvation treatment on the protein concentration in the mouthpart secretions of *Solanum nigrum*. Specifically: A is a comparative photograph of the feeding behavior of *Solanum nigrum* in the starvation treatment group and the untreated group; B is a comparative graph of the BCA colorimetric reaction between the starvation treatment group and the untreated group; C is a standard curve for BCA protein concentration determination in the starvation treatment group (x-axis: protein concentration mg / mL, y-axis: OD). 562 Absorbance value); D is the calculated protein concentration of the starvation treatment group (OS-4, OS-5). Detailed Implementation

[0026] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0028] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0029] Example 1: Collection and purification of secretions from the mouthparts of the small-flowered bug. 1. Preparation of an artificial feeding device for *Symplocos mingiensis* (1) Preparation of test insects: Collect 100-150 healthy small flower bugs (nymphs or adults) and transfer them to a sterile culture dish with a diameter of 6cm for later use.

[0030] (2) Pretreatment of sealing film: Cut a 3×3 cm Parafilm film and stretch it evenly in both directions until it can completely cover the opening of the culture dish (6 cm in diameter). Two intact stretch films are required for each set of devices.

[0031] (3) Primary sealing: Cover the opening of the culture dish with the first layer of extended film, ensuring that the edges are well sealed and the film surface is wrinkle-free, so as to prevent the insects from escaping.

[0032] (4) Construction of the feeding system: 200 μL of 2.5% (m / m) sucrose solution was evenly added to the surface of the primary sealing film in the form of small droplets. Then, a second layer of extended film was applied to form a sandwich structure, allowing the *Symplocos macrantha* to obtain nutrients by piercing and sucking the sucrose solution in the Parafilm sandwich. Finally, the petri dish was covered and inverted. A schematic diagram of the device structure is shown below. Figure 1 A.

[0033] 2. Collection and purification of secretions from the mouthparts of the small-flowered bug. (1) Collection of secretions: The small flower bugs were collected in an artificial feeding device and cultured for 24 h in an artificial climate incubator with 26 ± 1℃, 70 ± 5% relative humidity and complete darkness. Then, the sucrose solution in the Parafilm membrane interlayer was aspirated using an enzyme-free and sterile 1 mL syringe. This was the purified sample containing the mouthpart secretions of the small flower bugs.

[0034] (2) Pretreatment of ultrafiltration centrifuge tubes: Take 0.5 mL of sample loading volume in a 3 kDa ultrafiltration centrifuge tube, rinse thoroughly with Milli-Q H2O, and centrifuge at 4℃ and 9,000 rpm for 20 min to fully wet the filter membrane. Discard the filtrate, remove excess water, and store at 4℃ for later use.

[0035] (3) Sample concentration and purification: Transfer the sucrose solution collected in step (1) to a pre-cooled ultrafiltration centrifuge tube, centrifuge at 4℃ and 9,000 rpm for 18 min, and discard the filtrate from the outer tube. Replace with a new outer tube, invert the inner tube, centrifuge at 4℃ and 5,000 rpm for 2-3 min, and collect the concentrated solution in the inner tube to obtain a high-purity sample of *Mallotus maculatus* mouthpart secretions. See [link to sample collection and purification steps] for details. Figure 2 A.

[0036] (4) Protein concentration detection: A 2 mg / mL BSA protein standard solution was prepared using the BCA Protein Quantitative / Concentration Assay Kit (MeilunBio, China). As needed, BSA protein standard solutions were diluted with 2.5% sucrose solution to create gradient concentrations. The absorbance was measured at 562 nm, and a standard curve (R²>0.95) was plotted. 20 μL of the concentrated sample was taken, and the absorbance at 562 nm was measured. After subtracting the 2.5% sucrose solution blank control, the protein concentration of the sample was calculated based on the standard curve.

[0037] (5) Results The small flower bug can effectively feed on sucrose solutions in the interlayer of Parafilm membranes. This invention utilizes an artificial feeding device ( Figure 1 B) Collect mouthpart secretions from *Symplocos macrantha*. Behavioral observations show that both nymphs and adults of *Symplocos macrantha* can perform stable piercing-sucking behavior on the Parafilm membrane interlayer within the device. Figure 1 B). To further verify its feeding effectiveness, green fluorescent protein (GFP, 0.1 mg / mL) was mixed with 2.5% sucrose solution at a mass ratio of 1:10 as the feeding solution. Fluorescence detection results showed significant GFP signals in the digestive tract, excrement, and nymphs of adult *Symplocos mingiana*. Figure 1 (C) confirms that the artificial feeding system can effectively support the feeding needs of both nymphs and adults of the small flower bug.

[0038] Protein concentration test results of oral secretions Sucrose solutions were collected 24 h after feeding by *Symplocos macrantha*, and the protein concentration was determined after concentration. The results showed that the concentrated samples (OS-1, OS-2, OS-3) exhibited significantly enhanced BCA colorimetric reaction compared to the 2.5% sucrose solution (control). Figure 2 B). According to the standard curve ( Figure 2 C) Calculations show that the protein concentrations of OS-1, OS-2, and OS-3 are 0.4556 mg / mL, 0.5989 mg / mL, and 0.6863 mg / mL, respectively. Figure 2 D). This result indicates that the small flower bug releases mouthpart secretions during piercing and sucking feeding, and this method can efficiently capture the secreted proteins in these secretions.

[0039] Example 2: Effect of starvation treatment on mouthpart secretion collection efficiency 1. Experimental Methods After starving adult *Symplocos macrantha* for 1-2 days, an artificial feeding device was constructed according to the method in Example 1, and mouthpart secretions were collected and purified. The sucrose solution after feeding was collected, concentrated, and then the protein concentration was determined.

[0040] 2. Results The results showed that starvation treatment significantly improved the feeding induction efficiency of the artificial feeding system. Figure 3 A); The intensity of the BCA colorimetric reaction in the starvation-treated group samples (OS-4, OS-5) was significantly higher than that in the untreated group (OS-1, OS-2, OS-3). Figure 3 B); according to the standard curve ( Figure 3 C) Calculations showed that the protein concentrations in the starvation-treated group were 0.780 mg / mL (OS-4) and 0.799 mg / mL (OS-5), respectively, significantly higher than those in the untreated group. Figure 3D). These results indicate that starvation treatment can effectively promote the mouthpart secretion activity of the small flower bug, and that the feeding system of the present invention can efficiently capture the mouthpart secretion proteins released during piercing and sucking feeding.

[0041] 3. Conclusion Starvation treatment can effectively promote the secretory activity of the mouthparts of the small flower bug and improve the collection efficiency of protein in the secretions.

[0042] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for collecting and purifying the mouthpart secretions of the predatory natural enemy, the small-flowered bug, characterized in that... Includes the following steps: (1) Constructing an artificial feeding device: The device includes a container, a first membrane layer covering the opening of the container, and a second membrane layer disposed above the first membrane layer; a closed interlayer space is formed between the first membrane layer and the second membrane layer, and the interlayer space contains a nutrient solution that can be sucked by the stinger of the *Symplocos macrantha*; the test *Symplocos macrantha* is placed in the container; (2) The small flower bug pierces the first membrane layer to feed on the nutrient solution in the interlayer space. During this process, the small flower bug releases mouthpart secretions into the nutrient solution. The nutrient solution is collected to obtain a sample containing the mouthpart secretions. The nutrient solution is a sugar solution. (3) Place the sample collected in step (2) into an ultrafiltration centrifuge tube for centrifugation and concentration, discard the filtrate, then invert the inner tube of the ultrafiltration centrifuge tube for a second centrifugation, collect the concentrate, and obtain the purified oral secretion sample.

2. The method according to claim 1, characterized in that, In step (1), constructing the artificial feeding device specifically includes: (a) Preparation of test insects: Collect several healthy small flower bugs and transfer them to sterile petri dishes for later use; (b) Pretreatment of sealing film: Take Parafilm film and stretch it evenly in both directions until it can completely cover the opening of the culture dish. Two intact stretch films are required for each set of devices. (c) Primary sealing: The first layer of extended film is smoothly covered over the opening of the petri dish, ensuring that the edges are well sealed and the film surface is wrinkle-free, so as to prevent the insects from escaping. (d) Feeding system construction: Nutrient solution is evenly dripped onto the surface of the primary sealing film in the form of droplets, and then covered with a second layer of extended film to form a sandwich structure, so that the small flower bug can obtain nutrients by piercing and sucking the nutrient solution in the Parafilm membrane sandwich. Finally, the petri dish is covered and inverted.

3. The method according to claim 1, characterized in that, The sugar solution is a sucrose solution with a mass fraction of 1% to 5%.

4. The method according to claim 1, characterized in that, The tested *Mallotus cristatus* was starved before being placed in the container.

5. The method according to claim 4, characterized in that, The starvation treatment lasts for 12 to 48 hours.

6. The method according to claim 1, characterized in that, In step (2), the *Ming Xiaohua Bug* is cultured for 12 to 48 hours at 20–30°C, relative humidity of 50%–90%, in complete darkness or low light conditions.

7. The method according to claim 1, characterized in that, In step (3), the molecular weight cutoff of the ultrafiltration centrifuge tube is 1 to 10 kDa.

8. The method according to claim 1, characterized in that, In step (3), the conditions for centrifugal concentration are: temperature 4-10℃, rotation speed 8,000-12,000 rpm, and time 10-30 minutes; the conditions for secondary centrifugation are: rotation speed 3,000-8,000 rpm and time 1-5 minutes.

9. A sample of mouthpart secretions from the predatory natural enemy, the small-flowered bug, characterized in that... It is obtained by collecting and purifying using any one of claims 1 to 8.

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