Screening method and application of 4-hydroxyphenylacetic acid sex pheromone
By screening and isolating 4-hydroxyphenylacetic pheromone, and using it to confuse or lure male snails, the environmental friendliness and specificity of snails in the prior art are solved, and the effect of rapidly reducing population size is achieved.
Patent Information
- Application Number
- CN202310068414.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-06
- Publication Date
- 2025-08-12
- Estimated Expiration
- 2043-02-06
AI Technical Summary
It is difficult to effectively prevent and control snails with good life, and chemical control methods have negative impacts on the environment and lack strong specific and environmentally friendly control methods.
By screening and isolating 4-hydroxyphenylacetic pheromone, using it as sex pheromone to confuse or lure male snails, preventing mating behavior, metabolites were detected by ultrafiltration centrifugation and ultra-high performance liquid chromatography tandem mass spectrometry, and 4-hydroxyphenylacetic acid was screened as candidate pheromone.
It provides a green and effective prevention and treatment method for the Fushou Snail, which confuses male Fushou Snail by releasing 4-hydroxyphenylacetic pheromone, reduces mating reproduction rate, quickly reduces the population size, and is environmentally friendly.
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Figure CN116439173B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of ecological prevention and control, and in particular relates to a screening method for 4-hydroxyphenylacetic acid sex pheromone and an application thereof. Background Art
[0002] The golden apple snail (Pomacea canaliculata), also known as the golden apple snail, belongs to the genus Pomacea, family Ampullariidae, class Caenogastropoda. Native to freshwater basins in southern South America, it has now invaded Asia, Europe, and North America. Due to its high reproductive capacity, voracious feeding habits, and lack of effective natural enemies, it causes significant losses to aquatic plant cultivation and poses a serious threat to human health and life. Therefore, prevention and control of the golden apple snail is necessary to reduce agricultural losses, protect ecological balance, and safeguard human life.
[0003] The main methods for controlling golden apple snails at present include physical control, chemical control, agricultural control and biological control. It is almost impossible to achieve effective control by artificially capturing or physically isolating golden apple snails, and the cost is high. Although the use of pesticides, such as snailicide and metaldehyde, can eradicate golden apple snails in small water bodies, this chemical control method is not very specific and is likely to endanger other non-target organisms, such as other crustaceans, aquatic insects and fish. The negative impact of chemical pesticides on agricultural production and the ecological environment is becoming increasingly prominent. Therefore, it is very necessary to find a highly specific and environmentally friendly control method. Summary of the Invention
[0004] The object of the present invention is to provide a screening method for 4-hydroxyphenylacetic acid sex pheromone and its application to solve one of the above technical problems.
[0005] The present invention provides a method for screening 4-hydroxyphenylacetic acid sex pheromone, comprising the following steps:
[0006] Step 1: Prepare a heterosexual information source, collect sexually mature female and male golden apple snails, perform a biological activity assay on the heterosexual information source, and observe whether the golden apple snails exhibit tropism;
[0007] Step 2: Collect male golden apple snails to conduct a reaction experiment to the female information source before and after mating, and determine whether there is a difference in the selection of male golden apple snails when the female golden apple snails' metabolic feeding water before and after mating is used as the information source;
[0008] Step 3: Conduct behavioral responses of male golden apple snails to female information sources with molecular weights less than 5 KDa, 5-10 KDa, and greater than 10 KDa;
[0009] Step 4: Determine the composition of differential metabolites and screen for significantly differential metabolites;
[0010] Step 5: Six endogenous and non-toxic metabolites were screened out from the significantly different metabolites as candidate pheromones, and the behavioral characteristics of the candidate pheromones were determined to obtain the sex pheromone substance of the golden apple snail: 4-hydroxyphenylacetic acid.
[0011] The present invention also provides the use of the aforementioned 4-hydroxyphenylacetic acid to confuse or lure male golden apple snails. By releasing 4-hydroxyphenylacetic acid sex pheromones, male golden apple snails can be confused, thereby preventing them from seeking out female golden apple snails for mating. During the breeding season, the golden apple snail sex pheromones can also be used as an attractant to trap male golden apple snails.
[0012] The present invention also provides the use of the 4-hydroxyphenylacetic acid sex pheromone in preparing an attractant.
[0013] The principles and beneficial effects of the present invention are:
[0014] To address the problem of weak control of golden apple snails, the present invention provides a screening method for 4-hydroxyphenylacetic acid sex pheromone, which overcomes the problems of complex aquatic animal metabolites and difficult separation, breaking through the research bottleneck of golden apple snail sex pheromone screening. It has the advantages of a simple screening route and easy operation, providing a new, greener and more effective approach for the ecological control of golden apple snail populations, and laying a theoretical and technical foundation for the scientific prevention and control of the damage caused by golden apple snails.
[0015] The 4-hydroxyphenylacetic acid sex pheromone provided by the present invention is isolated from the pure water culture medium of female golden apple snails and purified using ultrafiltration centrifugation. Metabolites are detected using ultra-performance liquid chromatography-tandem mass spectrometry and confirmed by biological activity assays involving comparative analysis of standard compounds and pure water. During ecological control, the release of the 4-hydroxyphenylacetic acid sex pheromone can confuse male golden apple snails, thereby preventing them from seeking out females for mating. During the breeding season, the 4-hydroxyphenylacetic acid sex pheromone can also be used as an attractant to trap male golden apple snails. Once a large number of male individuals in a population are eliminated, the mating and reproduction rate can be effectively reduced, thereby rapidly reducing the population size. This biological control method is environmentally friendly, harmless to humans, and, in most cases, species-specific. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 Y-shaped flume for biological activity determination;
[0017] Figure 2 The behavioral response of the golden apple snail to the opposite-sex information source;
[0018] Figure 3 The behavioral responses of male golden apple snails to female information sources before and after mating;
[0019] Figure 4The behavioral responses of male golden apple snails to female information sources with different molecular weight cutoffs.
[0020] Figure 5 is a bar graph of the fold difference of differential metabolites;
[0021] Figure 6 is the Venn diagram of the differential metabolites among the groups;
[0022] Figure 7 Behavioral responses of male (A) and female (B) golden apple snails to different candidate cues;
[0023] Figure 8 It is the chemical structure of 4-hydroxyphenylacetic acid. DETAILED DESCRIPTION
[0024] The following is further described in detail through specific implementation methods:
[0025] Example 1
[0026] This example provides a method for screening 4-hydroxyphenylacetic acid sex pheromone, and the specific screening method is as follows:
[0027] Step 1: Experiment on the behavioral response of golden apple snails to opposite-sex information sources (Experiment 1)
[0028] 1.1 Preparing heterosexual information sources
[0029] The sex pheromones of the golden apple snail will be secreted into the surrounding water environment, so the water from the pond where the golden apple snail is raised is collected as a sample for extracting the sex pheromones.
[0030] During the breeding season of golden apple snails (April-October), 20 sexually mature female and male golden apple snails were collected from the wild and raised in a water tank. 0.5 L of pure water was added, and the pool water in the water tank was collected once every 3 hours, 4 times a day. The samples were combined and centrifuged (8000 g / min) to remove impurities. The supernatant was stored at -80°C to obtain a frozen sample, or vacuum freeze-dried to obtain a freeze-dried sample and then stored. For detection, 5 mL of pure water was used to dissolve the freeze-dried sample.
[0031] 1.2 Determination of the bioactivity of golden apple snails to opposite-sex information sources
[0032] The collected breeding pond water samples or separated samples are used as pheromone samples for biological activity testing to determine whether the test samples contain active pheromones.
[0033] like Figure 1The Y-shaped water tank shown in the figure includes a start arm and two bifurcated end arms. Sexually mature golden apple snails are placed at the end of the start arm. Pheromone samples (feeding water samples, separation samples, and standard chemicals) are absorbed by a sponge and randomly placed at the end of one of the end arms. Pure water is absorbed by a sponge at the end of the other end arm as a control. The golden apple snails are observed to see if they exhibit tropistic behavior.
[0034] To prevent apple snails from favoring one end arm over the other, the positions of the pheromone sample and pure water were swapped for every five snails in a group to ensure random selection of the two end arms. Behavioral experiments were conducted in a dark room or at night.
[0035] Selectivity was recorded 10 minutes after the golden apple snails adapted. Each experiment lasted 20 minutes, and each pheromone sample was repeated more than 50 times. The entire experimental process was recorded by a monitoring system to avoid human interference.
[0036] The researchers compared whether male and female golden apple snails showed behavioral differences in response to information sources of the opposite sex. A total of 120 golden apple snails were selected for the experiment, of which 70 male golden apple snails were used to test their behavioral responses to female information sources, and 50 female golden apple snails were used to test their behavioral responses to male information sources. For male golden apple snails, the number of snails that chose female information sources, pure water, and no response were 27, 14, and 29, respectively. Figure 2 For female golden apple snails, the number of snails that chose the male information source and pure water was 15 and 16 respectively, with a very small difference between the two.
[0037] The experimental data were tested by nonparametric chi-square test (Table 1). The results showed that female information sources had an attractive effect on male golden apple snails and the results were significant (χ 2 =4.122, df=1, P=0.042), while the male information source had no significant attraction effect on female golden apple snails (χ 2 =0.032, df=1, P=0.857), which indicates that the sexual attraction phenomenon of golden apple snails is a one-way sexual attraction of female snails to male snails.
[0038] Table 1 Chi-square test of the experiment on the behavioral response of golden apple snails to information sources of the opposite sex
[0039]
[0040]
[0041] Step 2: Experiment on the behavioral response of male golden apple snails to female information sources before and after mating (Experiment 2)
[0042] 2.1 Collection of female information sources before and after mating
[0043] Female golden apple snails that were raised alone in a water tank for more than 30 days and did not lay eggs were considered pre-mating apple snails; female golden apple snails that were raised together and completed mating within 5 days were considered post-mating apple snails.
[0044] Twenty female golden apple snails were taken before and after mating and raised in a water tank. Pure water was added to collect female information sources before and after mating.
[0045] 2.2 Determination of the biological activity of female information sources before and after mating
[0046] Using the behavioral responses of male golden apple snails as a benchmark, we determined whether male golden apple snails exhibited differences in their choices when using the metabolic feed water of female golden apple snails before and after mating as information sources. 100 male golden apple snails were selected for each behavioral response experiment, using the female information source before and after mating.
[0047] The results are as follows Figure 3 As shown, the number of snails that chose the female information source before mating, pure water, and no reaction were 45, 26, and 29, respectively. The number of snails that chose the information source after mating and pure water were 38 and 30, respectively, with a small difference between the two.
[0048] The experimental data were tested by nonparametric chi-square test (Table 2). The results showed that the female information source had an attractive effect on male golden apple snails before mating, and the results were significant (χ 2 =5.085, df=1, P=0.024), while the information source had no significant attraction effect on male golden apple snails after mating (χ 2 =0.941, df=1, P=0.332). This suggests that before mating, female golden apple snails release information substances to attract male golden apple snails for mating.
[0049] Table 2 Chi-square test of the behavioral response of golden apple snails to female information sources before and after mating
[0050] Information Source <![CDATA[χ 2 ]]> df P Pre-mating information sources 5.085 1 0.024* Post-mating information sources 0.941 1 0.332
[0051] Step 3. Experiment on the behavioral response of male golden apple snails to female information sources with different molecular weight cut-offs (Experiment 3)
[0052] 3.1 Collection of female information sources with different molecular weight cut-offs
[0053] Take an appropriate amount of 0.01g / L breeding water sample solution and place it in an ultrafiltration tube with a separation molecular weight cutoff value of 5KDa. Centrifuge and ultrafilter at 4°C. Add an appropriate amount of pure water to the upper part of the 5KDa ultrafiltration tube and transfer it to a 10KDa ultrafiltration tube. Ultrafilter and centrifuge again to obtain information source components with molecular weights less than 5KDa, 5-10KDa and greater than 10KDa.
[0054] 3.2 Bioactivity determination of female information sources with different molecular weight cut-offs
[0055] Using male golden apple snails as experimental subjects, a Y-shaped flume was used to compare the metabolic activity of females with molecular weights less than 5 kDa, 5-10 kDa, and greater than 10 kDa using rearing water as the information source to determine whether males differ in their selection. A total of 450 male golden apple snails were used in the selection experiment, and 150 samples were selected for the behavioral response experiments using females with molecular weights less than 5 kDa, 5-10 kDa, and greater than 10 kDa as the information source.
[0056] The experimental results are shown in Figure 4 The number of male golden apple snails that chose female information sources with molecular weight less than 5KDa and pure water were 42 and 52 respectively; the number of male golden apple snails that chose female information sources with molecular weight 5-10KDa and pure water were 64 and 41 respectively; and the number of male golden apple snails that chose female information sources with molecular weight greater than 10KDa and pure water were 58 and 46 respectively.
[0057] The experimental data were tested by nonparametric chi-square test (Table 3), which showed that female information sources with molecular weight of 5-10 KDa had an attractive effect on male golden apple snails, and the results were significant (χ 2 =5.038, df=1, P=0.025), and the molecular weight is less than 5KDa (χ 2 =1.064, df=1, P=0.302) and greater than 10 KDa (χ 2 =1.385, df=1, P=0.239) had no attraction to male golden apple snails, indicating that the effective attractant components were distributed in the 5-10 kDa retentate.
[0058] Table 3 Chi-square test of behavioral responses of male golden apple snails to female information sources with different molecular weight cut-offs
[0059] Information Source <![CDATA[χ 2 ]]> df P <5KDa 1.064 1 0.302 5-10 kDa 5.038 1 0.025* >10kDa 1.385 1 0.239
[0060] Step 4: Determination of differential metabolite composition (Experiment 4)
[0061] 4.1 Sample extraction
[0062] Frozen samples (male and female rearing water samples and their 5-10 kDa pheromone samples, and female pre- and post-mating pheromone samples and their 5-10 kDa pheromone samples) were slowly thawed at 4°C. After addition of a pre-chilled methanol / acetonitrile / water solution (2:2:1, v / v), the mixture was vortexed, sonicated at low temperature for 30 minutes, allowed to stand at -20°C for 10 minutes, and centrifuged at 4°C (14,000 g / min for 20 minutes). The supernatant was aspirated and dried under vacuum. For mass spectrometry analysis, 100 μL of acetonitrile-water solution (acetonitrile:water = 1:1, v / v) was added for reconstitution, vortexed, and centrifuged at 14,000 g at 4°C for 15 minutes. The supernatant was filtered through a 0.22 μm filter membrane before analysis.
[0063] 4.2 UPLC-MS / MS acquisition conditions
[0064] 4.2.1 Chromatographic conditions
[0065] Samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) system with a HILIC column; the column temperature was 25°C; the flow rate was 0.5 mL / min; the injection volume was 2 μL; the mobile phase composition was A: water + 25 mM ammonium acetate + 25 mM ammonia; B: acetonitrile.
[0066] The gradient elution program was as follows: 0-0.5 min, 95% B; 0.5-7 min, 95%-65% B; 7-8 min, 65%-40% B; 8-9 min, 40% B; 9-9.1 min, 40%-95% B; 9.1-12 min, 95% B.
[0067] 4.2.2 Q-TOF mass spectrometry conditions
[0068] The AB Triple TOF 6600 mass spectrometer (AB SCIEX) was used to collect the primary and secondary spectra of the samples:
[0069] Ion source: electrospray ionization (ESI) positive and negative ion source; ion source temperature: 600℃; nebulizer gas auxiliary heating gas 1 (Gas1): 60; auxiliary heating gas 2 (Gas2): 60; curtain gas (CUR): 30psi; spray voltage (ISVF) ±5500V (positive and negative modes); primary mass-to-charge ratio detection range: 60-1000Da, secondary product ion mass-to-charge ratio detection range: 25-1000Da, primary mass spectrometry scan accumulation time: 0.20s / spectra; secondary mass spectrometry scan accumulation time: 0.05s / spectra; secondary mass spectra were acquired using data-dependent acquisition mode (IDA) and peak intensity value screening mode; declustering voltage (DP): ±60V (positive and negative modes); collision energy: 35±15eV; dynamic exclusion of isotope ions range: 4Da.
[0070] 4.4 Screening and selection of differential metabolites
[0071] Non-targeted metabolome analysis was used to compare and screen the metabolites of samples from different experimental groups (Experiment 1: behavioral response experiment of golden apple snails to opposite-sex information sources; Experiment 2: behavioral response experiment of male golden apple snails to female information sources before and after mating; Experiment 3: behavioral response experiment of male golden apple snails to female information sources with different cutoff molecular weights; Experiment 4: determination of differential metabolite composition).
[0072] The male information source, post-mating information source, male 5-10KDa retentate and post-mating 5-10KDa retentate samples were used as control groups for multivariate statistical analysis. The strict orthogonal partial least squares discriminant analysis (OPLS-DA) was used to analyze the variables with the projection value VIP>1, P value<0.05 and log2 FC ||>1 (FC: fold difference) was used as the screening criterion for significant differential metabolites to screen metabolites with significant differential expression.
[0073] The histograms are used to visually display the up- and down-regulation of the significantly different metabolites screened between the groups in positive and negative ion modes ( Figure 5 ).
[0074] The results showed that a total of 27 significantly different metabolites were screened out in the pheromone samples of female and male feeding water, of which 7 were upregulated and 20 were downregulated; a total of 102 significantly different metabolites were screened out in the pheromone samples of female before and after mating, of which 94 were upregulated and 38 were downregulated; a total of 48 significantly different metabolites were screened out in the 5-10KDa pheromone samples of female and male, of which 19 were upregulated and 29 were downregulated; a total of 52 significantly different metabolites were screened out in the 5-10KDa pheromone samples of female before and after mating, of which 26 were upregulated and 26 were downregulated ( Figure 5The intersection of up-regulated differential metabolites among the groups was visually displayed using a Venn diagram ( Figure 6 ), and a total of 11 significantly differential metabolites were screened out, including 4-hydroxyphenylaceticacid, 1-monolinoleoyl-rac-glycerol, 4-imidazoleacrylic acid, Beta-estradiol, Ethyl p-coumarate, Oleic acid, L-saccharopine, Triethanolamine, 1-(benzo[d][1,3]dioxol-4-yl)-2-(methylamino)pentan-1-one, 3,4'-isopropylidenediphenol, and Met-Met-Arg (Table 4).
[0075] Table 4 Differential metabolites in the intersection between groups
[0076]
[0077]
[0078] Step 5: Determine the behavior of candidate information objects
[0079] Metabolites that were significantly upregulated and differentially expressed across the groups were identified as key metabolites for analysis. By reviewing literature and data, six endogenous, non-toxic metabolites were identified as candidate informants from the significantly differentially expressed metabolites: 4-hydroxyphenylacetic acid, 1-monolinoleoyl-rac-glycerol, 4-imidazoleacrylic acid, Beta-estradiol, Ethyl p-coumarate, and Oleic acid. Standard compounds were purchased and then subjected to biological activity assays.
[0080] The results showed that among the five differential metabolites selected, only 4-hydroxyphenylacetic acid could significantly attract male golden apple snails compared with the pure water control group (χ 2 =7.000, df=1, P=0.008*), but could not significantly attract female individuals (χ 2 =0.087, df=1, P=0.768). The other five metabolites had no significant attraction to either female or male individuals ( Figure 7 and Table 5). The results show that 4-hydroxyphenylacetic acid is the sex pheromone substance of golden apple snail. The chemical structure of 4-hydroxyphenylacetic acid is shown in Figure 8 .
[0081] Table 5 Chi-square test of the behavioral response of golden apple snails to different candidate information objects
[0082]
[0083]
[0084] Example 2
[0085] This example provides the use of 4-hydroxyphenylacetic acid sex pheromone in confusing or luring male golden apple snails.
[0086] For ecological control, 4-hydroxyphenylacetic acid pheromones can be released to confuse male golden apple snails, preventing them from seeking out females for mating. During the breeding season, 4-hydroxyphenylacetic acid pheromones can also be used as an attractant to trap male golden apple snails, achieving ecological control.
[0087] The present invention also provides the use of the aforementioned 4-hydroxyphenylacetic acid sex pheromone in the preparation of an attractant. 4-hydroxyphenylacetic acid can be used directly as an attractant or mixed with other auxiliary materials to prepare an attractant. The auxiliary materials can be used to extend the shelf life of 4-hydroxyphenylacetic acid, prolong the attractant duration of 4-hydroxyphenylacetic acid, and so on.
[0088] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.
Claims
1. The use of 4-hydroxyphenylacetic acid sex pheromone in confusing or luring male golden apple snails, characterized in that: 4-Hydroxyphenylacetic acid sex pheromone was obtained by screening using the following method: Step 1: Prepare a heterosexual information source, collect sexually mature female and male golden apple snails, perform a biological activity assay on the heterosexual information source, and observe whether the golden apple snails exhibit tropism; Step 2: Collect male golden apple snails to conduct a reaction experiment to the female information source before and after mating, and determine whether there is a difference in the selection of male golden apple snails when the female golden apple snails' metabolic feeding water before and after mating is used as the information source; Step 3: Conduct behavioral responses of male golden apple snails to female information sources with molecular weights less than 5 KDa, 5-10 KDa, and greater than 10 KDa; Step 4: Determine the composition of differential metabolites and screen for significantly differential metabolites; Step 5: Six endogenous and non-toxic metabolites were screened out from the significantly different metabolites as candidate pheromones, and the behavioral characteristics of the candidate pheromones were determined to obtain the sex pheromone substance of the golden apple snail: 4-hydroxyphenylacetic acid.
2. Use of the 4-hydroxyphenylacetic acid sex pheromone according to claim 1 in the preparation of an attractant.