Preparation method and application of apple snail slow-release lure core

Abstract

The application discloses a preparation method of an apple snail slow-release lure core and application thereof, and belongs to the technical field of green prevention and control of invasive harmful organisms. The preparation method comprises the steps of taking a hollow capillary as a slow-release carrier and encapsulating pheromone protein in the slow-release carrier. The amino acid sequence of the pheromone protein is shown as SEQ ID NO. 1. Through indoor and outdoor experiments, it is found that the prepared slow-release lure core realizes broad-spectrum trapping of female snails, male snails and medium snails based on the intraspecific specific recognition mechanism of the pheromone protein, avoids interference on non-target organisms such as fish and shrimps, and overcomes the defects of water failure and short effective period of traditional food lures. The application has simple preparation process, controllable cost, is suitable for green and precise prevention and control of apple snails in rice field and ditch and other aquatic crop production areas, and has wide popularization and application prospect.

Description

Technical Field

[0001] This invention relates to the field of green control technology for invasive pests, and in particular to a method for preparing a slow-release lure core for golden apple snails and its application. Background Technology

[0002] The golden apple snail (Pomacea canaliculata Lamarck) belongs to the family Pomacea, order Mesogastropoda, family Pomacea, class Gastropoda, phylum Mollusca. Also known as the giant apple snail or apple snail, it originated in the Amazon River basin of South America and was introduced to my country in the 1980s. It was among the first 16 major invasive alien species listed by the State Environmental Protection Administration (2002) and is also listed by the International Union for Conservation of Nature (IUCN) as one of the 100 most destructive invasive species globally. Golden apple snails lay their egg masses on the stems of rice, weeds, and other plants, as well as on surrounding ditches, walls, and field ridges. Both juvenile and adult snails bite and damage the main tillers and effective tillers of rice, leading to a reduction in effective panicles and thus yield loss, affecting the yield and quality of rice and other crops.

[0003] To mitigate water pollution, ecological damage, and resistance risks caused by chemical molluscicides, eco-friendly invasion control strategies have become a current research hotspot. Current physiochemical control techniques for *Pomacea canaliculata* rely on the application of attractants (mainly based on starch and yeast), but these methods are characterized by large dosages, easy water inactivation, short duration of effectiveness, and high cost. Furthermore, they also attract non-target organisms such as fish and shrimp in freshwater ecosystems, resulting in poor specificity. To address the challenges of controlling the invasive species *Pomacea canaliculata*, and based on the need for green biological control, this invention focuses on the preparation and application of slow-release attractants, using *Pomacea canaliculata* pheromones as the core. The products developed based on this invention can provide precise, specific, slow-release, and highly efficient green control products for aquatic crop production areas, possessing significant practical value and broad application prospects. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a slow-release lure for *Pomacea canaliculata* and its application, thereby addressing the problems existing in the prior art. This invention utilizes polymeric capillaries as a slow-release carrier to achieve long-term release of pheromone proteins; simultaneously, it leverages the species-specific recognition mechanism of pheromones to achieve precise attraction of adult male and female *Pomacea canaliculata* snails and intermediate snails, avoiding impact on non-target aquatic organisms. This is of great significance for achieving green control of *Pomacea canaliculata* in paddy fields and ditch habitats.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing a slow-release lure from the golden apple snail, comprising the steps of using a hollow capillary as a slow-release carrier and encapsulating an allergen protein within the slow-release carrier; wherein the amino acid sequence of the allergen protein is shown in SEQ ID NO.1.

[0006] Preferably, the preparation method includes the following steps: (1) Dissolve the freeze-dried allura protein powder in water to obtain a protein mother liquor; then mix the protein mother liquor with an organic solvent to prepare an allura protein core solution; wherein the organic solvent is prepared by papaya flavoring and ethyl dimethylbutyrate, or by mixing ethanol and water; (2) Cut the hollow capillary into a slow-release carrier of a predetermined length, then thermo-seal one end of the slow-release carrier, inject the core solution from the open end of the slow-release carrier, seal the open end, and freeze for storage.

[0007] Preferably, the volume of the sustained-release carrier injected into the lure solution is 15-30 μL, and the final concentration of allura protein in the lure solution is 4-20 μg / mL.

[0008] Preferably, the hollow capillary is made of polymer materials such as PE, PVC and TPU, with an outer diameter of 1.0-2.0 mm, an inner diameter of 0.5-1.0 mm, and a cut length of 5-8 mm.

[0009] Preferably, the organic solvent is prepared by mixing papaya flavoring and ethyl dimethylbutyrate in a volume ratio of 4:1, or by mixing 75% ethanol and water in a volume ratio of 1:4.

[0010] Preferably, the thermoplastic sealing conditions are: temperature 60-75 ℃, duration 5-10 s.

[0011] The present invention also provides a slow-release lure from the golden apple snail, which is prepared by the aforementioned preparation method.

[0012] The present invention also provides the application of the aforementioned slow-release lure core for golden apple snails in the preparation of a trapping device for controlling golden apple snails.

[0013] The present invention also provides a golden apple snail trapping device, comprising a trapping cage and a slow-release lure for golden apple snails disposed within the trapping cage.

[0014] The present invention also provides a method for attracting golden apple snails, wherein the slow-release lure core or the golden apple snail attracting device is placed in a field ditch containing golden apple snails to attract them.

[0015] The present invention discloses the following technical effects: (1) The present invention is based on the preparation of lure cores by the pheromone protein of golden apple snail. By utilizing the high intraspecificity of pheromones, it can achieve broad-spectrum attraction of female snails, male snails and medium snails, avoiding interference with non-target organisms such as fish and shrimp, and has good ecological safety.

[0016] (2) This invention uses a capillary slow-release carrier and effectively controls the release rate by optimizing the material and sealing process. The indoor T-maze test showed that 23 snails were attracted in 24 hours, which was significantly higher than the control group. The outdoor ditch test showed that the lure core had a duration of 14 days. On the 7th day, 67 small snails and 27 adult snails were captured, while only 5 snails were captured in the control group. This overcomes the defects of traditional lures that are ineffective when exposed to water and have a short duration of effect.

[0017] (3) The preparation process of this invention is simple and the cost is controllable. It can reduce the use of chemical molluscicides and is suitable for green prevention and control in paddy fields and ditches and other aquatic crop production areas. It has broad prospects for promotion. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 To slow-release the inducing core product; Figure 2 For the T-maze activity test in the slow-release decoy chamber (24h); A: Schematic diagram of the T-maze setup; B: Actual diagram of the T-maze; Figure 3 Evaluation of the effectiveness of lures in outdoor ditch habitats; A: Placement diagram of lures in outdoor ditch habitats; B: Number of snails trapped by lures at different time points; Figure 4 A: Number of snails trapped on day 7 after the lure was placed in the ditch habitat; B: Number of snails trapped on day 7 in the experimental cage; C: Number of snails trapped on day 7 in the control cage; D: Statistical results of the number of snails trapped on day 7 in the experimental and control cages. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1: A method for preparing a slow-release lure from the golden apple snail S1: Allergen protein recombinant synthesis Based on the sequence of the golden apple snail pheromone protein (NCBI Public Database Accession No. XP_025099450.1, SEQ ID NO.1), the active protein was recombinantly synthesized using the pET23b vector and a cell-free expression system of E. coli. After purification and verification (the synthesis and purification methods refer to the inventor's patent application "CN 202510106170.6 A method for recombinant expression and purification of golden apple snail aggregation pheromone protein"), the pheromone protein was freeze-dried to obtain lyophilized powder.

[0026] >XP_025099450.1 temptin-like isoform X1 [Pomacea canaliculata]: MLYNVTVCTLLLTFSQEDFRRRMASLTVLASILLVCLLPLALPFPTFQDSIPNGKNVVNPCDKSPWPGVGHQASQGGGPRNPFGLDFAAAGNAWTTALCQKDSDGDGVSNGEELGDSSCQWNATNPVNLASPKGHPGICEPVGSAKCGNSWFACPS (155aa).

[0027] S2: Production of Slow-Release Lure Core TPU capillaries with an outer diameter of 1.65 mm and an inner diameter of 0.76 mm were selected, and the capillaries were cut into slow-release cores with a length of 5 mm.

[0028] S3: Preparation of the core-lubricating solution Take 20 μg of allura protein lyophilized powder from S1, add 50 μL of ddH2O, centrifuge at 5000 rpm for 2 min at 4 ℃, and after it is fully dissolved, take 12.5 μL of allura protein solution and mix it thoroughly with 500 μL of organic solvent (the organic solvent consists of 400 μL of papaya flavoring + 100 μL of dimethyl butyrate) to prepare the lure solution; the concentration of allura protein in the lure solution is 10 μg / mL.

[0029] S4: Core Pouring and Encapsulation A small heat sealer (Aonian) was used to seal one end of the sustained-release carrier in S2 at a temperature of 60 ℃ for 10 s. Using a 10 μL microsyringe (Micro Sample Syringe, Sangon Biotech (Shanghai) Co., Ltd.), approximately 15 μL of the prepared solution from S3 was injected into one end of the core opening (enough to fill the cavity of the sustained-release carrier). Finally, the sealing operation was repeated to completely seal one end of the opening, and the solution was stored at -20℃ for later use.

[0030] Example 2: A method for preparing a slow-release lure core from a golden apple snail S1: Allergen protein recombinant synthesis Based on the sequence of the golden apple snail pheromone protein (NCBI Public Database Accession No. XP_025099450.1, SEQ ID NO.1), the active protein was recombinantly synthesized using the pPICZα A vector and the Pichia pastoris expression system. After purification and verification (the synthesis and purification methods refer to the inventor's patent application "CN 202510106170.6 A method for recombinant expression and purification of golden apple snail aggregation pheromone protein"), the pheromone protein was freeze-dried to obtain lyophilized powder.

[0031] S2: Production of Slow-Release Lure Core PE capillary tubes with an outer diameter of 1.2 mm and an inner diameter of 0.6 mm were selected, and the capillary tubes were cut into slow-release cores with a length of 6 mm.

[0032] S3: Preparation of the core-lubricating solution Take 20 μg of allura protein lyophilized powder from S1, add 50 μL of ddH2O, centrifuge at 5000 rpm for 2 min at 4℃, and after it is fully dissolved, take 10 μL of allura protein solution and mix it thoroughly with 500 μL of organic solvent (the organic solvent consists of 400 μL of papaya flavoring + 100 μL of dimethylbutyrate) to prepare the lure solution; the concentration of allura protein in the lure solution is 8 μg / mL.

[0033] S4: Core Pouring and Encapsulation One end of the sustained-release carrier in S2 was sealed using a small heat sealer (Aonian) at a temperature of 70 ℃ for 8 s. Using a 10 μL microsyringe (Micro Sample Syringe, Sangon Biotech (Shanghai) Co., Ltd.), approximately 20 μL of the prepared solution from S3 was injected into one end of the core opening (enough to fill the cavity of the sustained-release carrier). Finally, the sealing process was repeated to completely seal one end of the opening, and the solution was stored at -20℃ for later use.

[0034] Example 3: A method for preparing a slow-release lure from the golden apple snail S1: Same as S1 in Example 1; S2: Select a PVC capillary tube with an outer diameter of 2.0 mm and an inner diameter of 1.0 mm. Cut the capillary tube into a slow-release core with a length of 8 mm.

[0035] S3: Preparation of the core-lubricating solution Take 20 μg of allura protein lyophilized powder from S1, add 50 μL of ddH2O, centrifuge at 5000 rpm for 2 min at 4 ℃, and after it is fully dissolved, take 25 μL of allura protein solution and mix it thoroughly with 500 μL of organic solvent (the organic solvent consists of 100 μL of 75% ethanol + 400 μL of ddH2O) to prepare the lure solution; the concentration of allura protein in the lure solution is 20 μg / mL.

[0036] S4: Core Pouring and Encapsulation A small heat sealer (Aonian) was used to seal one end of the sustained-release carrier in S2 at a temperature of 75 ℃ for 5 s. Using a 10 μL microsyringe (Micro Sample Syringe, Sangon Biotech (Shanghai) Co., Ltd.), approximately 30 μL of the prepared solution from S3 was injected into one end of the core opening (enough to fill the cavity of the sustained-release carrier). Finally, the sealing process was repeated to completely seal one end of the core, and the solution was stored at -20℃ for later use.

[0037] Experimental Example 1: Indoor Activity Verification of Sustained-Release Decoction Core 1. Experimental location Laboratory of Shanghai Institute of Agricultural Sciences and Ecological Environment Protection.

[0038] 2. Experimental Methods Slow-release lures were prepared according to Example 1. Three lures were placed into one black bait ball (commercially available; to better utilize the slow-release effect, a small opening or hole was cut or punched in the sealed end of the lure before inserting it into the bait ball), and placed on the right arm of the T-maze apparatus. A white centrifuge tube containing 2 mL of ddH2O was placed on the left arm as a blank control. Fifty snails, including adult and juvenile snails, were collected from the field and placed at one end of the long arm of the T-maze. The snails and lures were submerged in 2-3 L of water throughout the apparatus. After 24 hours, the number of snails selected for the blank control and those with pheromone lures was counted.

[0039] like Figure 2 As shown, the results revealed that after 24 hours, 23 golden apple snails (8 males, 6 females, and 9 medium-sized snails) were selected from the right arm of the lure, significantly more than the control group (4 adult snails). Therefore, the lure prepared according to the method of this invention has a good effect and exhibits significant attraction activity for golden apple snail populations indoors. Preliminary evidence suggests that this lure has no difference in attraction effect on female and male snails, possessing dual attraction for both sexes, and exhibits a significant aggregation effect on medium-sized snails.

[0040] Experiment Example 2: Outdoor Activity Verification of Sustained-Release Decoction Core 1. Experimental location The rice paddies at the Shanghai Academy of Agricultural Sciences Grain and Soil Experiment Station were surveyed from July 3 to 17, 2026.

[0041] 2. Experimental Methods Slow-release lures were prepared according to Example 1. Six lures were divided into two black bait balls (three lures in each ball; to better utilize the slow-release effect, a small opening or hole was cut or punched at the sealed end of the lure before inserting it into the bait ball). The lures were then assembled and placed together into a modified eel trap (6 openings) as the experimental trap. No lures were placed in the control trap. Both the experimental and control traps were placed together in the ditch along the edge of the rice paddy and secured with rope. The traps were retrieved 6 hours after placement and on days 3, 7, 11, and 14, and the number of golden apple snails trapped in the traps was counted.

[0042] The results showed that after 14 days, the total number of golden apple snails harvested from the lure cages was 168, significantly more than the control cage (24 snails), demonstrating that the lure cages had a better slow-release effect and longer duration of action. Figure 3 Furthermore, on day 7, the number of golden apple snails in the experimental cage was significantly higher than that in the control cage, with 27 adult snails and 67 small and medium-sized snails, while only 5 snails were harvested from the control cage. This demonstrates that the slow-release lure of the present invention has a significant aggregation effect on small and medium-sized snails, and its activity is highest on day 7. Figure 4 ).

[0043] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a slow-release lure core from a golden apple snail, characterized in that, The method includes the steps of using a hollow capillary as a sustained-release carrier and encapsulating allergen protein within the sustained-release carrier; wherein the amino acid sequence of the allergen protein is shown in SEQ ID NO.

1.

2. The preparation method according to claim 1, characterized in that, The preparation method includes the following steps: (1) Dissolve the freeze-dried allura protein powder in water to obtain a protein mother liquor; then mix the protein mother liquor with an organic solvent to prepare an allura protein core solution; wherein the organic solvent is prepared by papaya flavoring and ethyl dimethylbutyrate, or by mixing ethanol and water; (2) Cut the hollow capillary into a slow-release carrier of a predetermined length, then thermo-seal one end of the slow-release carrier, inject the core solution from the open end of the slow-release carrier, seal the open end, and freeze for storage.

3. The preparation method according to claim 2, characterized in that, The volume of the sustained-release carrier injected into the lure solution is 15-30 μL, and the final concentration of allura protein in the lure solution is 4-20 μg / mL.

4. The preparation method according to claim 2, characterized in that, The hollow capillary tube is made of polymer materials such as PE, PVC and TPU, with an outer diameter of 1.0-2.0 mm, an inner diameter of 0.5-1.0 mm, and a cut length of 5-8 mm.

5. The preparation method according to claim 2, characterized in that, The organic solvent is prepared by mixing papaya flavoring and ethyl dimethylbutyrate in a volume ratio of 4:1, or by mixing 75% ethanol and water in a volume ratio of 1:

4.

6. The preparation method according to claim 2, characterized in that, The conditions for the thermoplastic seal are: temperature 60-75 ℃, duration 5-10 s.

7. A slow-release lure core from a golden apple snail, characterized in that, It is prepared by the preparation method described in any one of claims 1-6.

8. The application of the slow-release lure core of golden apple snail as described in claim 7 in the preparation of a trapping device for controlling golden apple snails.

9. A snail-attracting device, characterized in that, The snail trap includes a trap cage and a slow-release lure core as described in claim 7, which is disposed within the trap cage.

10. A method for attracting golden apple snails, characterized in that, The slow-release lure core of golden apple snails as described in claim 7 or the golden apple snail attracting device as described in claim 9 is placed in the water of a field ditch containing golden apple snails to attract them.

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