Inhibitor for inhibiting development of ampullaria gigas eggs and application thereof

Abstract

The invention discloses an inhibitor for inhibiting development of ampullaria gigas eggs and application of the inhibitor. Through medicament screening, a compound mixture of salad oil and acetic acid provided by the invention can effectively inhibit the development of ampullaria gigas egg masses, the optimal ratio of salad oil to acetic acid is 10: 1, and 0.07 g of medicament is applied to 1 g of eggs. Laboratory and field tests show that the inhibition rate of the mixture to ampullaria gigas egg masses reaches 100%. A scanning electron microscope is used for observing the processed ampullaria gigas ovum structure to show that more pores are formed on the surfaces of the ovum masses after the mixture is processed, so that the mixture can enter an embryo easily. Further respiratory action experiments show that the respiratory action of the egg masses treated by the mixture is obviously reduced within 24 hours, which is speculated that the mixture permeates into the egg masses, the respiratory action of the egg masses is inhibited, and the development of the eggs is further inhibited. The invention provides an environment-friendly, efficient and cheap technical means for prevention and treatment of ampullaria gigas.

Description

Technical Field

[0001] The invention belongs to the field of biological invasion prevention and control, and relates to an inhibitor for inhibiting the development of golden apple snail eggs and an application thereof. Background Art

[0002] The golden apple snail (Pomacea canaliculata), also known as the snow snail, apple snail, golden apple snail, and large bottle snail, belongs to the class Gastropoda of the phylum Mollusca and is a freshwater organism originating from the Amazon River basin in South America. Initially, golden apple snails were introduced to Asia for food and ornamental purposes due to their high protein content. However, due to their poor taste, market demand was insufficient or they escaped into the wild, leading to a surge in their populations and flooding in natural waters, causing significant damage to crops, especially rice, and seriously affecting agricultural production. 1 Due to the lack of effective control, the golden apple snail has become increasingly rampant in recent years. Faced with the severe situation of the spread of golden apple snails, finding effective prevention and control methods has become particularly urgent.

[0003] The golden apple snail looks like a field snail, with male and female members separated and reproducing through cross-breeding. Eggs are usually laid at night on dry objects or plants above the water surface. The first-born golden apple snail eggs are relatively soft, and the surface is covered with a gelatinous mucus, which forms a layer of film after drying and solidifying. The eggs are oval, about 2mm in diameter, and bright red in color. The number ranges from dozens to thousands. After hatching, the young golden apple snails fall into the water and begin to prey. Usually, a female snail can produce more than 325,000 young snails in two generations a year, and its reproductive capacity is extremely strong. 2 The rampant golden apple snails not only cause huge damage to crops, especially rice, seriously affecting agricultural production, but also destroy the balance of the ecological environment.

[0004] Currently, physical, biological and chemical methods can be used to deal with the infestation of golden apple snails.

[0005] (1) Physical methods

[0006] In rice fields, manually removing eggs and adult snails is an effective control method, but it is labor-intensive and time-consuming. To increase farmers' enthusiasm, many regions have implemented a "purchase + resource utilization / harmless treatment" strategy, where the government purchases snail eggs and then centrally processes or utilizes them as resources. 3 Taking advantage of the habit of golden apple snails to lay eggs on land, bamboo poles can be inserted into rice fields to attract them to lay eggs and destroy them. 4 Because small snails are more resistant to water flow, while large snails easily migrate with the water flow, potentially affecting surrounding wetlands, irrigation blocking techniques can be used, such as setting up nets and snail-killing bags at water inlets and outlets, to control golden apple snails in rice fields and prevent the invasion of foreign snails.

[0007] (2) Biological methods

[0008] Biological control is an effective method for controlling golden apple snails, but the number of natural enemies is often insufficient. Therefore, it is particularly important to introduce carp, black carp and Chinese soft-shelled turtle for all-weather control. 5 In the Philippines, red fire ants can effectively destroy apple snail eggs and reduce their numbers. The predatory behavior of natural enemies can also disrupt the life cycle of apple snails. 6 Poultry and birds, especially ducks and egrets, are also natural enemies of golden apple snails. Rice-duck farming can effectively reduce the number of golden apple snails. 7 The control effect of Chinese soft-shelled turtle is particularly significant. In addition, the feeding preference of golden apple snail on duckweed helps to reduce the damage to rice. Foreign studies have shown that low-cost biological resources and agricultural waste, such as garlic peels and coffee grounds, have the potential for biological control. 8 Plant-based snailicides are a safe and environmentally friendly method of prevention and control. For example, tea saponins in tea bran have a strong killing effect on golden apple snails. 9 Further research found that ragweed 10 , Alternanthera philoxeroides 11 , Tung oil tree 12 , oleander 13 Various plant extracts can also effectively prevent and control golden apple snails.

[0009] (3) Chemical methods

[0010] Chemical control is still the main means of controlling golden apple snails due to its rapid and low-cost advantages. Commonly used snailicides include metaldehyde granules and snailicide ethanolamine salt. 14 However, over-reliance on chemical pesticides may disrupt ecological balance and biodiversity, so it is recommended to use them in combination with other sustainable measures and avoid using them in aquatic animal farming areas to prevent pollution. Some chemical molluscicides are highly toxic to non-target organisms such as fish and crustaceans. 15 Oxidative molluscicides based on ozone and peroxides offer an environmentally friendly alternative. A mixture of copper chelates and ozone water is effective against golden apple snails, but further research is needed to improve its application efficiency. 16 A mixture of vegetable oil, camphor oil, and emulsifier is effective in killing apple snail eggs, but the compound ingredients are harmful to the environment, and the mixture is viscous and difficult to spray, which is not conducive to large-scale use. 17 .

[0011] Regarding the current methods of snail control, it can be seen that the reason why the spread of golden apple snails has not been effectively controlled is mainly due to two reasons: (1) low efficiency; (2) toxicity and harm and not suitable for promotion.

[0012] By analyzing and comparing the properties and effects of numerous snail-killing agents, the inventors have proposed a novel control strategy. This strategy specifically targets the eggs of golden apple snails, based on a core concept: preventing the eggs from hatching can effectively curb the rapid spread of golden apple snails. Through careful material selection, repeated formulation, and numerous experiments, the inventors have ultimately developed an innovative method and formulation for inhibiting the hatching of golden apple snail eggs, without any potential safety hazards to the ecological environment or human health. The inventors hope that this method will play a more comprehensive and effective role in the control of golden apple snails. Summary of the Invention

[0013] The present invention aims to address the deficiencies in the prior art by proposing an environmentally friendly method for killing golden apple snail eggs, comprising a compound containing salad oil and acetic acid as its main components. The method has been demonstrated in laboratory and field trials to be completely effective in killing golden apple snail eggs, exhibiting excellent resistance to rainwater erosion and effectively preventing the hatching of egg masses with a single application. Furthermore, while controlling the eggs, this method can reduce the extinction-prone impact on adult snail populations, thereby maintaining biodiversity to a certain extent. The environmentally friendly, efficient, and cost-effective method for controlling golden apple snails proposed by the present invention can be applied to agricultural production and ecological protection.

[0014] The invention provides an inhibitor for inhibiting the development of golden apple snail eggs. The inhibitor comprises salad oil and acetic acid.

[0015] Preferably, the volume ratio of salad oil to acetic acid in the inhibitor is 2-20:1.

[0016] Further preferably, the volume ratio of salad oil to acetic acid in the inhibitor is 10-20:1.

[0017] Further preferably, the volume ratio of salad oil to acetic acid in the inhibitor is 2:1.

[0018] Further preferably, the volume ratio of salad oil to acetic acid in the inhibitor is 5:1.

[0019] Further preferably, the volume ratio of salad oil to acetic acid in the inhibitor is 20:1.

[0020] Further preferably, the volume ratio of salad oil to acetic acid in the inhibitor is 10:1.

[0021] The present invention also provides a medicine / pharmaceutical composition comprising the inhibitor as described above and a pharmaceutically acceptable carrier.

[0022] Furthermore, the drug / drug composition is used alone.

[0023] Preferably, the pharmaceutically acceptable carrier refers to a drug that does not produce adverse, allergic, or other untoward reactions when properly administered to an animal. Pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers. These substances are used as needed to help stabilize the formulation or to enhance the activity or its bioavailability or to produce an acceptable taste or odor when administered orally. Preferably, the drug / drug composition may further contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc.

[0024] Preferably, the dosage forms of the drug / pharmaceutical composition include injections, sterile powders for injection, tablets, pills, capsules, lozenges, elixirs, powders, granules, syrups, solutions, tinctures, aerosols, powder sprays, or suppositories. The drugs / pharmaceutical compositions in the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0025] Specifically, the medicine / drug composition includes liquid dosage forms, gaseous dosage forms, solid dosage forms, semi-solid dosage forms, and the like.

[0026] The drug / pharmaceutical composition can be in various dosage forms conventional in the art, preferably in the form of solid, semi-solid, gaseous or liquid, i.e., an aqueous solution, non-aqueous solution or suspension, more preferably a tablet, capsule, granule, injection or infusion, etc. Preferably, the drug / pharmaceutical composition can also be administered as an aerosol or coarse spray.

[0027] The present invention also proposes the use of the inhibitor, or the drug / drug composition in inhibiting the development of golden apple snails and killing golden apple snail eggs.

[0028] The application proceeds as follows:

[0029] (1) Mixing salad oil and acetic acid solution;

[0030] (2) Pour the prepared compound into a spray bottle;

[0031] (3) Before using the spray bottle, shake the liquid in the bottle to ensure that the salad oil and acetic acid are evenly mixed;

[0032] (4) For the golden apple snail egg masses in the fields or ditches, directly point the spray bottle at the golden apple snail egg masses and spray them.

[0033] In step (1), the volume ratio of the salad oil to the acetic acid solution is 2:1, 5:1, 10:1, or 20:1; preferably, it is 10:1.

[0034] In step (4), the dosage of spraying is 0.07g compound / 1g apple snail eggs. As long as the surface of the eggs is covered with the agent, the eggs can be killed. It is not advisable to spray too much. The experimental results show that for 1dm 2 Applying 2.86 mL of the drug to the plant will cause its death.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] Highly efficient killing: The present invention adopts a compound of salad oil and acetic acid. Experimental results show that at the optimal ratio of 10:1, the inhibition rate of golden apple snail eggs reaches 100%, which is far superior to the existing snail-killing drugs such as calcium chloride and tea saponin.

[0037] Cost-effectiveness: Compared with existing hydrophilic agents, the salad oil compounded with 10% acetic acid selected in the present invention is not only relatively cheap and easy to use, but also can effectively reduce the egg hatching rate to 0, showing a higher cost-effectiveness.

[0038] Environmentally friendly: The ingredients of the compound of the present invention are all edible and harmless to humans. Since it can effectively kill eggs at relatively low concentrations, it is harmless to surrounding plants and animals (safety testing has been performed). Compared with existing snail-killing agents, it reduces environmental pollution. Furthermore, due to its fat-soluble nature, it is not easily affected by rainwater erosion, maintaining its killing effect.

[0039] Easy to operate: The compound of the present invention is easy to atomize and spray, and is not too viscous even at high concentrations, thereby optimizing the spraying effect and making the operation easier.

[0040] The mechanism is relatively clear: through observation by scanning electron microscopy and Raman spectroscopy, the compound of the present invention may affect the metabolic process of the egg by destroying the calcium carbonate structure on the surface of the eggshell, thereby blocking embryonic development.

[0041] In summary, the present invention shows more significant beneficial effects than the existing technology in terms of killing efficiency, cost-effectiveness, environmental friendliness, ease of operation and clear mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1: The inhibitory effect of the compound pesticide on the eggs of the golden apple snail. d: day, the number of days after the golden apple snail egg mass was treated with the pesticide.

[0043] Figure 2 : Field experiment. a, 90-m ditch; b, egg mass of the control group (10 days); c, egg mass of the experimental group (10 days).

[0044] Figure 3 Egg masses dropped into the water after salad oil treatment. a, Egg masses dropped into the water in the control group; b, Unhatched eggs in the control group (fished out of the water after 3 days); c, Egg masses dropped into the water in the oil-sprayed group; d, Unhatched eggs in the experimental group (fished out of the water after 3 days); e, Juvenile snail development in the control group; f, Juvenile snail development in the experimental group.

[0045] Figure 4 SEM and Raman spectroscopy observation and analysis of the eggshell surface. a&b, control group, a (300x), b (3000x); c&d, experimental group, c (300x), d (3000x). e, Raman spectroscopy observation of the eggshell surface.

[0046] Figure 5 : Respiratory rate was measured at different time points after spraying. Con is the water control group.

[0047] Figure 6 :Using low-field nuclear magnetic resonance to study the changes of water molecules in eggs, Figure 6 a is the change of water molecules during normal development of snail eggs; Figure 6 b shows the changes in water molecules in snail eggs after application of pesticides.

[0048] Figure 7 :SOD enzyme activity detection and transcriptome analysis diagram, Figure 7 a represents the loss of SOD enzyme activity as the drug continues to act; Figure 7 b: After 4 days of drug treatment, the expression levels of PcSOX9L, HSP70, and Cur-Zn·SOD genes in the golden apple snail egg mass were significantly reduced compared with the control group; Figure 7 c is a diagram showing the relationship between genes and cellular energy metabolism, lipid metabolism, material transport, and material degradation processes; Figure 7 d and e show that after 4 days of drug treatment, the expression levels of 168 genes in the cells were significantly decreased, and the expression levels of another 58 genes were significantly upregulated. DETAILED DESCRIPTION

[0049] The present invention is further described in detail with reference to the following specific examples and accompanying drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0050] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention, its application, or use. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0051] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0052] The present invention proposes an environmentally friendly and highly effective method for inhibiting the development of golden apple snail egg masses. Through agent screening, we found that a mixture of salad oil and acetic acid effectively inhibits the development of golden apple snail egg masses. The optimal ratio was 10:1 salad oil:acetic acid, with 0.07g of the agent applied to 1g of eggs. Both laboratory and field trials demonstrated that the mixture achieved a 100% inhibition rate on golden apple snail egg mass. Scanning electron microscopic observation of the structure of golden apple snail eggs after treatment revealed that the mixture formed numerous pores on the egg surface, facilitating the entry of the mixture into the embryo. Further respiration experiments revealed a significant decrease in respiration within 24 hours after treatment. This is presumably due to the mixture penetrating the egg mass, inhibiting respiration and, in turn, egg development. This invention provides an environmentally friendly, highly effective, and inexpensive method for the prevention and control of golden apple snails.

[0053] Example 1 Preliminary screening of molluscicidal agents

[0054] After reviewing the literature, the present inventors conducted preliminary laboratory experiments with hydrophilic agents, but found that they did not adhere well to the surface of golden apple snail eggs. Given the hydrophobic nature of golden apple snail eggs, various fat-soluble substances were tested (Table 1): ethanol, emulsifiers, and other types of vegetable oils, animal oils such as fish oil, and mineral oils such as silicone oil, applied individually and in combination.

[0055] Table 1

[0056]

[0057] The results show that the combination of acetic acid and salad oil is the best, and its advantages are as follows:

[0058] (1) Spraying only acetic acid can kill 100% of the insects, but it has a strong pungent odor and causes significant damage to animals, plants and other environmental organisms, and also pollutes the inorganic environment such as soil;

[0059] (2) Although the killing rate can reach 100% when salad oil (including other vegetable oils) is used alone, the atomization effect of the oil is poor, the amount used is difficult to control, and the amount is often excessive and uneven. Therefore, it is not recommended to use it alone;

[0060] (3) The combination of acetic acid and salad oil is relatively cheap and has a 100% killing rate. If other substances are added, it will increase the environmental pollution burden and the cost. Therefore, the present invention recommends the use of a 10:1 mixture of salad oil and acetic acid.

[0061] (4) In summary, the combination of acetic acid and salad oil has the effect of 1+1>2. On the one hand, acetic acid reacts with calcium carbonate on the surface of the egg shell, destroying the egg shell structure. The gaps formed may facilitate the entry of oil into the egg fluid and disrupt the normal development of the embryo. On the other hand, the mixture of acetic acid and salad oil effectively enhances the atomization effect of the oil, allowing it to be sprayed evenly and controllably.

[0062] Example 2: Salad oil combined with acetic acid can enhance the killing efficiency of golden apple snail eggs

[0063] According to the preliminary screening results of Example 1 of the present invention, the present invention explores the optimal compound ratio of salad oil and acetic acid. The results show that when the ratio of salad oil to acetic acid is 2:1, the development rate of golden apple snail eggs is 11.94%; when the ratio is increased to 5:1, the development rate drops to 2.16%. When the salad oil concentration is further increased to 10:1, the inhibition rate reaches 100%, that is, the development rate drops to 0. Although the inhibition rate remains 100% when the ratio reaches 20:1, the present invention observes that the viscosity of high-concentration salad oil increases, affecting its atomization spraying effect. Therefore, taking into account the inhibitory effect and ease of use, the present invention recommends that the optimal ratio of salad oil to acetic acid is 10:1. This ratio can not only effectively inhibit the development of golden apple snail eggs, but also optimize the spraying effect (Table 2).

[0064] Table 2

[0065]

[0066] Note: The drug ratio is the ratio of salad oil to acetic acid.

[0067] In an experiment using a combination of salad oil and acetic acid to inhibit the development of golden apple snail eggs, the present invention observed a significant phenomenon: the treated golden apple snail eggs remained stable, maintaining a bright red color, while the untreated control group eggs gradually changed color from bright red to pink and grayish white as they developed, and eventually hatched into small snails. This observation indicates that the combination of salad oil and acetic acid can affect the pigment metabolism process of golden apple snail eggs, blocking their normal embryonic development ( Figure 1 ).

[0068] Example 3: The actual application effect in the field is remarkable

[0069] In order to verify whether the killing effect of salad oil and acetic acid compound on golden apple snail eggs in the laboratory is also effective under field conditions, the present invention conducted a field test in the Jiangsu and Zhejiang regions where golden apple snails are seriously infested. A 90-meter-long ditch was selected and divided into two sections, each about 45 meters ( Figure 2 ).

[0070] One section of the ditch was treated with a mixture of salad oil and acetic acid, while another section was sprayed with an equal amount of water as a control. The control ditch contained 98 egg masses, approximately 10,000 eggs, of which 89 egg masses, approximately 9,000 eggs, were successfully hatched, with a hatching rate of 90.8%. The experimental group had 102 egg masses, approximately 10,400 eggs, but only 5 eggs from one egg mass were successfully hatched, with a hatching rate close to 0 (Table 3). This result confirms that the combination of salad oil and acetic acid is equally effective in killing golden apple snail egg masses in the wild, providing a new green approach for the prevention and control of golden apple snails that is simple to operate, highly targeted, and environmentally friendly.

[0071] Table 3

[0072]

[0073] In addition, the present invention also proposes that the compound of salad oil and acetic acid can directly inhibit the development of the eggs after being applied to the eggs of the golden apple snail, and its effect is not affected by rain erosion. The egg masses are immediately put into water after being sprayed with medicine, and the development of the egg masses is observed. The experimental results show that the golden apple snail egg masses in the control group can continue to develop after falling into the water, with a hatching rate of 41.9%. The hatching rate of the golden apple snail egg masses in the oil-sprayed group after falling into the water is 6.5%, but the hatched young snails do not have the ability to continue to develop into adult snails (Table 4). This is different from the effect of the snail-killing drugs such as calcium chloride and tea saponin mentioned in the prior art that are easily washed away and thus become ineffective. The compound of the present invention can effectively resist rain erosion and maintain its killing effect, thereby avoiding the reduction in effect due to environmental factors ( Figure 3 ).

[0074] Table 4

[0075]

[0076] Example 4 Toxicology Experiment

[0077] In response to the potential phytotoxicity risks of the present invention during precise application around water bodies and in fields, the present invention evaluates its environmental safety through theoretical calculations and actual exposure analysis, combined with the determination of the maximum lethal dose of plants.

[0078] (1) Determination of the maximum lethal dose of plants and analysis of theoretical exposure dose

[0079] The drug dosage parameters involved in this experiment are: 2.86mL / m 2(Threshold), 286mL / m 2 (100% lethal concentration, LC 100 The test plant was Ophiopogon bodinieri H.Lév., and the exposure method was foliar spraying, with a concentration of 2.86 mL / m 2 , 286mL / m 2 The actual application parameters are as follows: the application rate for a single target (egg) is 0.143 mL / block, and the theoretical maximum egg density per unit area is 1905 eggs / m 2 , the theoretical maximum demand is calculated to be 1905 pieces / m 2 ×0.143mL / piece≈272mL / m 2 It can be found that the theoretical maximum dosage (272mL / m 2 ) close to LC 100 (286mL / m 2 ), although actual exposure is much lower than the theoretical value (see below for details).

[0080] (2) Actual exposure correction

[0081] The precise application of pesticides is reflected in the fact that the pesticide is only sprayed in small amounts on the egg masses, rather than covering a large area. The spray deviation rate is: >90% of the pesticide falls on the surface of the target animal, making the dose directly contacting non-target animals extremely low. Regarding the estimation of actual exposure dose, the non-target organism exposure dose is <10% of the theoretical value (i.e. <27.2mL / m 2 ), which is much lower than LC 100 (286mL / m 2 ), which is also far below the threshold (2.86 mL / m 2 ) is more than 10 times.

[0082] In summary, foliar spraying offers precise application, primarily targeting the target species with minimal impact on non-target animals. Actual exposure during actual application was significantly lower than the theoretical value, and exposure to non-target organisms was low, demonstrating the safety of this application method, which effectively targets the target species with minimal risk to non-target organisms.

[0083] Example 5 Study on the mechanism of salad oil and acetic acid compound inhibiting the development of golden apple snail eggs

[0084] (1) Observation of eggshell surface structure

[0085] When deeply exploring the mechanism of the inhibitory effect of the compound of salad oil and acetic acid on the development of apple snail egg masses, the present invention used a scanning electron microscope (SEM) to observe and analyze the apple snail egg shells after drug treatment. The experimental results showed that the surface of the apple snail egg shells in the untreated group had uniform protrusions, while the surface of the egg shells in the experimental group was uneven and had many pores. This phenomenon may be attributed to the chemical reaction between the acetic acid in the compound drug and the calcium carbonate component on the surface of the egg shell, thereby destroying the structure of calcium carbonate. The egg shell components were further analyzed by Raman spectroscopy, and the results showed that there was almost no difference in the spectral profiles of the experimental group and the control group, which indicated that the composition of the egg shell did not change, but the content of the corresponding chemical components in the egg shell did change, which may be the key reason for inhibiting the development of apple snail eggs ( Figure 4 ).

[0086] (2) Analysis of egg respiration

[0087] The present invention uses infrared spectroscopy to measure CO2, and tests the respiratory rate of golden apple snail egg masses at different time points after drug treatment to evaluate the effect of the salad oil and acetic acid compound on egg respiration. The experimental results show that within 16 hours after drug application, the respiratory rate of the egg masses did not differ significantly from that of the control group. However, one day after drug application, the respiratory rate of the egg masses dropped to about 50% of that of the control group. Further observation showed that after four days of drug application, the respiratory rate of the golden apple snail egg masses sprayed with the drug almost completely stopped ( Figure 5 This phenomenon indicates that the snail eggs do not stop breathing immediately after drug spraying, but gradually stop breathing over time, accompanied by the cessation of development.

[0088] Based on the above experimental results, the present invention infers that the salad oil-acetic acid compound may mainly destroy the calcium carbonate component on the surface of the egg mass, allowing the oil and acetic acid molecules to enter the egg, thereby interfering with the normal metabolic function of the cell and ultimately inhibiting the development process of the egg. Further observation found that under normal circumstances, the egg mass of the golden apple snail is initially bright red. During the development process, its color will gradually change from bright red to pink until it becomes grayish white, and then the small snails will hatch. However, the red color of the egg mass after the drug treatment is not only brighter, but also remains unchanged throughout the growth and development stage, and no small snails can be hatched. Given that the bright red color of the golden apple snail eggs is caused by carotenoids in the cells 18 The phenomenon that the egg mass remains bright red after application of the pesticide is speculated to be due to the salad oil-acetic acid compound entering the egg and affecting the metabolic process of carotenoids, thereby inhibiting the normal development of the egg mass.

[0089] (3) Low-field NMR to study changes in water molecules in eggs

[0090] In order to further explore the changes in water molecules in the eggs of the golden apple snail after drug treatment, the present invention uses low-field nuclear magnetic resonance to explore the changes in water molecules in the eggs. Using hydrogen as a probe, the development process of the golden apple snail was observed non-destructively. The results showed that there were two peaks. The apex (100-200ms) was free water, representing the water in the cells or intercellular matrix of the golden apple snail eggs; the apex at (5-20ms) was bound water, which was water bound to biological macromolecules. When the snail eggs develop normally, the peak gradually shifts to the right over time, the water molecules become more active, and differentiates into two peaks at 192h ( Figure 6 After application, the peak shifted to the left with the development time, the activity of water molecules decreased, and no differentiation was observed at 192h ( Figure 6 , b). The signal intensity is weaker than that of normally developing snail eggs, and water loss is more rapid.

[0091] When golden apple snail eggs hatch normally, the fertilized eggs likely undergo mitosis to form multicellular embryos. The cell number continues to increase, forming a blastocyst, which then develops into a gastrula, and finally, into different tissues and organs. During this process, the activity of free water in the golden apple snail gradually increases. As different tissues form, the free water breaks down into free water and immobile water, with relaxation times of 500-1000ms and 50-100ms, respectively. Although the sealing film prevents water loss during this process, it continues to be consumed, resulting in a decrease in the overall peak area.

[0092] The present invention thus speculates that spraying the agent inhibits the normal development of the golden apple snails, preventing the initiation of cell mitosis, resulting in rapid water loss, a continuous decrease in free water activity (biological fluidity) in the body, a reduction in relaxation time, and a decrease in peak area.

[0093] (IV) SOD enzyme activity detection and transcriptome analysis

[0094] From a molecular biological perspective, based on the above experimental results, the present inventors speculate that the direct cause of the stagnation of egg mass development may be the gradual cessation of respiration, leading to death. So, what factors exactly cause this inhibition of respiration? The present inventors speculate that this may be related to changes in the expression of related proteins and genes caused by the compound drug after entering the cell.

[0095] First, the present invention measured the activity of superoxide dismutase (SOD) in the egg mass. Experimental data showed that in the early stage of spraying (8 hours), the SOD enzyme activity in the egg mass increased by 1.6 times compared with the control group, showing a slight upward trend; 16 hours after spraying, the SOD enzyme activity dropped to 80% of the control group; 48 hours later, it dropped to 60% of the control group; and 4 days later, the SOD enzyme activity was almost zero. This result shows that after the egg mass is treated with the compound, the SOD enzyme activity will increase in the short term, which may be the feedback regulation of the cells to the increased concentration of free radicals in the body. As the drug effect continues, the intracellular oxidation level further increases, and excessive free radicals eventually lead to the loss of SOD enzyme activity ( Figure 7 , a).

[0096] To further explore the changes in gene expression, the present invention analyzed the functions of important genes involved in the development of golden apple snail eggs. The SSH9 gene encodes the PcOvo1 protein, a glycolipid carotenoid protein that absorbs visible light and resists light radiation. The bright red color of the golden apple snail egg mass is due to the binding of the PcOvo1 protein to carotene. 19 The PcSOX9L gene may play a key role in the early embryonic development and sex determination of golden apple snails. 20 The protein encoded by the HSP70 gene is one of the heat shock proteins with relatively high content in the cytoplasm. It has ATPase activity and can use the energy provided by ATP to promote the correct folding of proteins. It participates in the folding, transport, assembly and degradation of proteins, prevents protein aggregation and promotes the repair of damaged proteins. 21 The protein encoded by the Cu-Zn SOD gene has antioxidant function 20 .

[0097] Using real-time fluorescence quantitative PCR technology, the present invention found that after 4 days of drug treatment, the expression levels of PcSOX9L, HSP70, and Cu-Zn SOD genes in the golden apple snail egg mass were significantly reduced compared with the control group ( Figure 7 , b), indicating that drug treatment can inhibit key genes in the development of golden apple snail eggs, leading to cell death. Notably, among the numerous genes, the expression of the SSH9 gene increased significantly, consistent with our observation that the bright red color of golden apple snail egg masses became more pronounced after drug application. The present inventors speculate that this may be because the oil in the compound extracts the carotene from the PcOvo1 protein after entering the cells, prompting the cells to upregulate the expression of the SSH9 gene, thereby increasing the synthesis of the PcOvo1 protein through a negative feedback mechanism.

[0098] To further clarify the overall changes in gene expression in cells of golden apple snail eggs after treatment with salad oil-acetic acid mixture, the present invention carried out cell transcriptome analysis. The experimental results showed that after 4 days of drug treatment, the expression levels of 168 genes in the cells were significantly decreased, and the expression levels of another 58 genes were significantly increased ( Figure 7 , d, e). Analysis of these significantly changed genes through KEGG signaling pathways revealed that a considerable number of genes were significantly related to cellular energy metabolism, lipid metabolism, material transport, and material degradation processes, with the differences in energy metabolism being particularly significant ( Figure 7 , c).

[0099] Example 6: A method for killing golden apple snail eggs using a mixture of salad oil and acetic acid. The following are the specific steps for implementing the method:

[0100] 1. Raw material preparation:

[0101] Purchase edible salad oil and acetic acid. Ensure the quality and purity of the raw materials to ensure the effectiveness and safety of the compound.

[0102] 2. Preparation of compound:

[0103] Mix salad oil and acetic acid solution in a 10:1 volume ratio. Specifically, measure 10 parts salad oil in a measuring container, then add 1 part acetic acid solution, stirring thoroughly to ensure the two are completely mixed.

[0104] 3. Load the spray bottle:

[0105] Place the prepared compound into a spray bottle. Choose a spray bottle with good atomization to ensure a uniform mist during spraying, which will improve the killing effect.

[0106] 4. Preparation before use:

[0107] Before using the spray bottle, be sure to shake the liquid in the bottle to ensure that the salad oil and acetic acid are evenly mixed to ensure the killing effect.

[0108] 5. Spraying operation:

[0109] For golden apple snail egg masses found in fields or ditches, simply aim the spray bottle at the egg mass and spray. For each 2g egg, only 0.14g of the agent is needed. With good atomization, only one spray is needed to effectively kill the golden apple snail egg mass.

[0110] In order to demonstrate the superiority of the present invention, the following comparative examples are provided:

[0111] Comparative Example 1:

[0112] Tea saponin, a common molluscicide on the market, was sprayed using the same experimental procedures as in Example 6 of the present invention, and the hatching rate and killing efficacy of the egg masses were recorded. Comparative results showed that the killing rate of the compound of the present invention was 100%, while tea saponin achieved the best killing rate of 80% (but was easily washed away by rainwater and had biotoxicity), significantly outperforming common molluscicides on the market.

[0113] Comparative Example 2:

[0114] Different ratios of salad oil and acetic acid were used for spraying, and the killing effect at different ratios was recorded. Experimental data showed that a ratio of 10:1 achieved the best balance between killing effect and spraying operation, as shown in Table 2 of the present invention.

[0115] Comparative Example 3:

[0116] The compound of the present invention was sprayed under different environmental conditions (such as different humidity and temperature), and the killing effect was recorded. The experimental results show that the compound of the present invention can maintain a stable killing effect under different environmental conditions, showing good environmental adaptability. The results show that the hatching rate of the golden apple snail egg masses after falling into the water in the clear water control group was 41.9%, while the hatching rate of the egg masses treated with the compound of the present invention was only 6.5%, and the hatched young snails could not continue to develop into adult snails. There is a significant difference in the effect compared with traditional snail-killing drugs such as calcium chloride and tea saponin, as shown in Table 4.

[0117] The advantages of the present invention are fully demonstrated by the above examples and comparative examples. The present invention is not only simple to operate, but also can effectively, economically and environmentally solve the problem of killing golden apple snail egg masses, and has broad application prospects.

[0118] References

[0119] 1. Li Xuxia et al. Study on the distribution status, damage and control strategies of golden apple snail. Jiangxi Science 41, 236-243+265, doi:10.13990 / j.issn1001-3679.2023.02.004(2023).

[0120] 2. Ren He, Gao Hongjuan, Xue Juan, Gong Ning & Chen Xiang. Biological characteristics and control of golden apple snails. Bulletin of Biology 55, 1-3 (2020).

[0121] 3. Zha Guoxian, Sun Zhenjun, Yang Pingjun, Gao Huihui. Study on the occurrence pattern of golden apple snail and farmland control technology in Suzhou City[J]. Plant Quarantine, 2022, 36(04):82-85.

[0122] 4. Chu Shaoyuan, Zhang Jiaen, Guo Jing, Chen Hao, Tan Yihua. Effects of different rod insertion methods on attracting golden apple snails to lay eggs in rice fields[J]. Journal of South China Agricultural University, 2018, 39(05): 39-46.

[0123] 5. Dong S, Zheng G, Yu

[0124] 6. Abdullah T, GassaA, Aminah SN, AnnisaaN W. The population of Solenopsis sp. (Hymenoptera: Formicidae) in four conditions of rice held embankment[J]. IOPConference Series: Earth and Environmental Science, 2021:807.

[0125] 7. Yang Shuying et al. An ecological method for controlling golden apple snails in rice fields using tea seed cake and a two-stage rice field duck farming system. Invention patent CN 109938165A. 2019.

[0126] 8. Khalisanni K. Effect of organic waste extracts as potential biocontrol of golden apple snail[J]. Journal of Tropical Plant Physiology, 2020, 12(2): 1-7.

[0127] 9. He J.Control effect of tea bran on Pomacea canaliculata[J].JilinAgriculture, 2014,(8):14-15.

[0128] 10、Huang R.The prevention and control effect of ragweed extract onPomacea canaliculata and its active components[D].HunanAgriculturalUniversity,2017.

[0129] 11、Z G,Tan J C,Liu J.Experimental Study on Poisoning of Pomaceacanaliculata by Saponin from Alternanthera philoxeroides[J].Shanghai Journalof Agriculture,2011,27(1):80-83.

[0130] 12、Wang Z Y,Du Y Q,Cen Y Z.Pharmacodynamic test of methanol extractfrom leaves of Jatropha Curcas[J].Journal of Southern Medical University,2009,29(6):1235-1237.

[0131] 13、Dong D Q,Chen J M,Yu X P,Chen L Z.Evaluation oftoxic effectsofdifferent solvent extracts ofNeriumindicum on apple snails[J].ZhejiangJournal ofAgriculture,2009,21(2):154-158.

[0132] 14、Huang Z C,Long C Y,Yu Q S,Cheng S F,Zhong F J,Li J M,Ou HM.Control effects offour common molluscicides against Pomaceacanaliculata inpaddy fields[J].World Pesticides,2023,45(06):56-60.

[0133] 15. Attademo A, Lajmanovich R, Peltzer P. Acute toxicity of metaldehydein the invasive rice snail Pomacea canaliculata and sublethal effects ontadpoles of a non-target species (Rhinella arenarum) [J]. Water Air SoilPollution, 2016, 227: 400-409.

[0134] 16. Zhang W T. Preparation and antibacterial activity of tea saponin from Camellia oleifera shell[D]. Hainan University, 2019.

[0135] 17. Chen Zhenliu. A method and composition for inhibiting the hatching of golden apple snail eggs. Patent document TW200735772A. 1995.

[0136] 18. Dreon, MS, Heras, H., & Pollero, RJ (2006). Biochemical composition, tissue origin and functional properties of egg perivitellins from Pomaceacanaliculata. BIOCELL, 30(2), 359-365.

[0137] 19. Zhou, H. et al. Identification of putative virulence-associated genes of Haemophilus parasuis through suppression subtractivehybridization. Veterinary microbiology 144, 377-383, doi:10.1016 / j.vetmic.2010.01.023(2010).

[0138] 20. Sun, J. et al. First Proteome of the Egg Perivitelline Fluid of aFreshwater Gastropod with Aerial Oviposition. Journal of Proteome Research 11, 4240-4248 (2012).

[0139] 21. Song, HMet al. Molecular characteristics of the HSP70gene and its differential expression in female and male golden apple snails (Pomaceacanaliculata) under temperature stimulation. Cell stress & chaperones 19, 579-589, doi:10.1007 / s12192-013-0485-0 (2014).

[0140] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0141] As used in the present invention, the term "comprising" is an open expression, that is, including the contents specified in the present invention, but not excluding other aspects.

[0142] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0143] Although the embodiments of the present disclosure have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations may be made to the embodiments without departing from the principles and spirit of the present disclosure, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An inhibitor for inhibiting the development of golden apple snail eggs, characterized in that The inhibitor comprises salad oil and acetic acid.

2. The inhibitor according to claim 1, characterized in that The volume ratio of salad oil to acetic acid in the inhibitor is 2-20:

1.

3. The inhibitor according to claim 2, characterized in that The volume ratio of salad oil to acetic acid in the inhibitor is 10-20:

1.

4. The inhibitor according to claim 3, characterized in that The volume ratio of salad oil to acetic acid in the inhibitor is 20:

1.

5. The inhibitor according to claim 3, characterized in that The inhibitor further comprises salad oil and acetic acid in a volume ratio of 10:

1.

6. A drug / drug composition, characterized in that The drug / pharmaceutical composition comprises the inhibitor according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier.

7. The drug / pharmaceutical composition according to claim 6, characterized in that The drug / drug composition can be used alone or in combination with other drugs.

8. Use of the inhibitor according to any one of claims 1 to 5 or the drug / drug composition according to claim 6 or 7 in inhibiting the development of golden apple snail eggs or in killing golden apple snail eggs.

9. The use according to claim 8, characterized in that The application comprises the following steps: (1) Mixing salad oil and acetic acid solution; (2) Pour the prepared compound into a spray bottle; (3) Before using the spray bottle, shake the liquid in the bottle to ensure that the salad oil and acetic acid are evenly mixed; (4) For the golden apple snail egg masses in the fields or ditches, directly point the spray bottle at the golden apple snail egg masses and spray them.

10. The use according to claim 9, characterized in that In step (1), the volume ratio of the salad oil to the acetic acid solution is 10:1, and the spraying amount is 0.07 g of the compound per 1 g of golden apple snail eggs.

Images