Application of a new sex pheromone mestranol

Abstract

The present invention belongs to the technical field of ecological control, and specifically discloses the application of a new sex pheromone mestranol in confusing or luring male Procambarus clarkii. The new sex pheromone mestranol provided by the present invention overcomes the problems of complex and difficult separation of aquatic animal metabolites, breaks through the research bottleneck of Procambarus clarkii sex pheromone screening, provides a more green and effective new way for the ecological control of Procambarus clarkii population, and lays a theoretical and technical foundation for scientific prevention and control of the harm of Procambarus clarkii. During ecological control, the purpose of confusing male Procambarus clarkii can be achieved by releasing the new sex pheromone mestranol, thereby preventing them from looking for female Procambarus clarkii for mating. In the breeding season, the new sex pheromone mestranol can also be used as an attractant to trap male Procambarus clarkii. Once a large number of male individuals in the population are eliminated, the mating and reproduction rate can be effectively reduced, thereby achieving the purpose of rapidly reducing the population size.

Description

Technical Field

[0001] The invention belongs to the technical field of ecological prevention and control, and in particular relates to the application of a new sex pheromone, mestranol. Background Art

[0002] Procambarus clarkii belongs to the genus Procambarus, belonging to the phylum Arthropada, class Crustacea, order Decapoda, family Cambaridae, commonly known as freshwater crayfish. Procambarus clarkii is native to North America and is distributed all over the world due to its strong vitality, wide adaptability and strong reproductive capacity. It is an extremely invasive alien invasive species. Once introduced, Procambarus clarkii will become a competitive wild dominant species in the local ecological environment, seriously endangering local species, reducing local biodiversity, and having a great impact on the local ecological environment. Therefore, in ecologically fragile areas, wetland reserves and other ecosystems, it is necessary to prevent and control Procambarus clarkii to protect the ecological environment.

[0003] At present, the main methods for controlling Procambarus clarkii are artificial capture, physical isolation and chemical control. Chemical control can use pesticides, such as cypermethrin and deltamethrin, to eradicate Procambarus clarkii in small water bodies, but it is not specific and easily endangers other non-target organisms, such as other crustaceans, aquatic insects and fish, and has an increasingly negative impact on agricultural production and the ecological environment. The sex pheromone of Procambarus clarkii is currently unknown, and it is impossible to use sex pheromones to carry out biological control research. Therefore, it is very necessary to find a new sex pheromone, mestranol, that is highly specific and environmentally friendly for biological control. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a new sex pheromone mestranol to solve the above technical problems.

[0005] The invention provides a new sex pheromone mestranol for use in confusing or luring male Procambarus clarkii.

[0006] The present invention also provides the use of the novel sex pheromone mestranol in preparing an attractant.

[0007] The present invention also provides a method for screening the novel sex pheromone mestranol, comprising the following steps:

[0008] Step 1, collecting sexually mature female and male Procambarus clarkii to perform biological activity determination on opposite-sex information sources, and observing whether Procambarus clarkii individuals show tropism behavior;

[0009] Step 2, behavioral response experiment of male Procambarus clarkii to female information sources with molecular weight less than 5KDa, 5-10KDa and greater than 10KDa;

[0010] Step 3, preparing active fragments of sex pheromones to narrow the screening range of target compounds;

[0011] Step 4, determining the composition of differential metabolites and screening for significantly differential metabolites;

[0012] Step 5: Screen out endogenous and non-toxic metabolites from the significantly different metabolites as candidate information substances, measure the behavior of the candidate information substances, and obtain the sex pheromone substance of Procambarus clarkii: mestranol.

[0013] The principles and beneficial effects of the present invention are:

[0014] The control of Procambarus clarkii is weak, the sex pheromone of Procambarus clarkii is currently unknown, and it is impossible to use sex pheromone to carry out biological control research. The mestranol provided by the present invention overcomes the problems of complex and difficult separation of aquatic animal metabolites, breaks through the research bottleneck of Procambarus clarkii sex pheromone screening, provides a greener and more effective new way for the ecological control of Procambarus clarkii populations, and lays a theoretical and technical foundation for the scientific prevention and control of the harm of Procambarus clarkii.

[0015] During ecological control, the mestranol provided by the present invention can confuse or lure male crayfish by releasing the new sex pheromone mestranol, preventing male crayfish from looking for female crayfish for mating. In the breeding season, mestranol can be used as an attractant to trap male crayfish. Once a large number of male crayfish individuals in the population are eliminated, the mating and reproduction rate can be effectively reduced, thereby achieving the purpose of rapidly reducing the population size. This biological control method is environmentally friendly, harmless to the human body, and has the advantages of species specificity in most cases.

[0016] The mestranol sex pheromone provided by the present invention is separated from the pure water breeding liquid of female Procambarus clarkii, separated and purified by ultrafiltration centrifugation, and metabolites are detected by ultra-high performance liquid chromatography tandem mass spectrometry, and determined by a biological activity determination method of comparative analysis of standard compounds and pure water. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a longitudinal cross-sectional view of a Y-shaped water tank;

[0018] Figure 2 Chromatogram of the 5-10 kDa fragment of male and female separated by semi-preparative HPLC (A) female; (B) male;

[0019] Figure 3 Prepare fragment metabolite classifications for Procambarus clarkii PFF6 and PFM6;

[0020] Figure 4The figure shows the expression fold difference of the significant difference metabolites in positive ion (A) and negative ion (B) modes;

[0021] Figure 5 The figure shows the attraction effect of different concentrations of mestranol on male Procambarus clarkii. DETAILED DESCRIPTION

[0022] The following is further described in detail through specific implementation methods:

[0023] Example 1: A new sex pheromone mestranol

[0024] The specific screening methods are as follows:

[0025] Step 1: Experiment on the behavioral response of Procambarus clarkii to information sources of the opposite sex (Experiment 1)

[0026] 1.1 Preparing heterosexual information sources

[0027] Female Procambarus clarkii excretes sex pheromones along with urine into the surrounding water environment through the nephrolithiasis. Therefore, in this experiment, the water from the pond where Procambarus clarkii was raised was collected as a sample for extracting sex pheromones.

[0028] From April to October (the breeding season of Procambarus clarkii), 1000 sexually mature female and male crayfish were collected and raised separately. Every 20 crayfish were raised in a water tank. 0.5L of pure water was added. The pool water in the tank was collected once every 3 hours, and 4 times a day. The samples were combined and centrifuged (8000g / min) to remove impurities. The supernatant was stored at -80℃ to obtain a frozen sample, or vacuum freeze-dried to obtain a freeze-dried sample and then stored. For detection, 5mL of pure water was used to dissolve the freeze-dried sample.

[0029] 1.2 Determination of the biological activity of Procambarus clarkii to the opposite sex information source

[0030] The collected pool water samples or separated samples are used as pheromone samples for biological activity testing to determine whether the test samples contain active pheromones.

[0031] Firstly, a Y-shaped tank was used to compare whether there were behavioral differences between male and female Procambarus clarkii in response to information sources of the opposite sex.

[0032] like Figure 1 The Y-shaped water tank shown in the figure includes a start arm and two forked end arms. Sexually mature Procambarus clarkii are placed at the end of the start arm, and pheromone samples (rearing 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 to observe whether the Procambarus clarkii individuals show tropism behavior.

[0033] In order to avoid the preference of Procambarus clarkii for the terminal arm on one side, the positions of pheromone samples and pure water were swapped in every group of 5 Procambarus clarkii to ensure that the choice of Procambarus clarkii for the two terminal arms was random.

[0034] Selectivity was recorded 5 minutes after the crayfish adapted. Each experiment lasted 5 minutes, and each pheromone sample was repeated more than 100 times. The entire experimental process was recorded by a monitoring system to avoid human interference.

[0035] Compare whether there are behavioral differences between male and female crayfish to opposite-sex information sources. A total of 280 crayfish were selected for the experiment, of which 160 male crayfish were selected for the behavioral response experiment of male crayfish to female information sources; 120 female crayfish were used for the behavioral response experiment of female crayfish to male information sources. For male crayfish, the number of shrimps that chose female information sources and pure water was 69 and 37, respectively (Table 1). For female crayfish, the number of shrimps that chose male information sources and pure water was 35 and 37, respectively, and the difference between the two was very small.

[0036] The experimental data were subjected to nonparametric chi-square test (Table 1). The results showed that female information sources had an attractive effect on male Procambarus clarkii and the results were significant (χ 2 =9.66, df=1, P<0.01), while the male information source had no significant attraction to female Procambarus clarkii (χ 2 =0.06, df=1, P=0.814), which indicates that the sexual attraction phenomenon of Procambarus clarkii is a unidirectional sexual attraction of female shrimp to male shrimp.

[0037] Table 1 Chi-square test table of the experimental behavior response of Procambarus clarkii to information sources of the opposite sex

[0038]

[0039] Step 2: Experiment on the behavioral response of male Procambarus clarkii to female information sources with different molecular weight cutoffs (Experiment 2)

[0040] 2.1 Collection of female information sources with different molecular weight cut-offs

[0041] 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.

[0042] 2.2 Determination of the biological activity of female information sources with different molecular weight cut-offs

[0043] Using male Procambarus clarkii as the experimental object, the metabolism of female Procambarus clarkii with molecular weight less than 5KDa, 5-10KDa and greater than 10KDa was compared with the information source of rearing water, and a Y-shaped tank was used to determine whether there was a difference in selection among male Procambarus clarkii. A total of 300 male Procambarus clarkii were used in the selection experiment, and 100 Procambarus clarkii samples were selected for the behavioral response experiment of female information sources with molecular weight less than 5KDa, 5-10KDa and greater than 10KDa.

[0044] The experimental results are shown in Table 2. The numbers of male Procambarus clarkii that chose female information sources with a molecular weight less than 5KDa and pure water were 42 and 52, respectively; the numbers of male Procambarus clarkii that chose female information sources with a molecular weight of 5-10KDa and pure water were 55 and 28, respectively; and the numbers of male Procambarus clarkii that chose female information sources with a molecular weight greater than 10KDa and pure water were 28 and 29, respectively.

[0045] The experimental data were tested by nonparametric chi-square test (Table 2), which showed that female information sources with molecular weight of 5-10 KDa had an attractive effect on male Procambarus clarkii, and the results were significant (χ 2 =8.78, df=1, P<0.01), and the molecular weight was less than 5KDa (χ 2 =0.26, df=1, P=0.611) and greater than 10 KDa (χ 2 =0.02, df=1, P=0.895) had no attraction to male Procambarus clarkii, indicating that the effective attraction components were distributed in the 5-10 KDa retentate.

[0046] Table 2 Chi-square test of the behavioral responses of male Procambarus clarkii to female information sources with different molecular weight cut-offs

[0047]

[0048] Step 3: Isolation of the active fragments of sex pheromones

[0049] Weigh 1g of the 5-10kDa female crayfish sex pheromone active fragment dry product, add 1L of ultrapure water to fully dissolve it to make a stock solution (stock solution concentration: 1g / L). Set the semi-preparative HPLC experimental parameters: mobile phase ratio of methanol / ultrapure water (60 / 40, v / v); wavelength: 210nm and 254nm; single injection volume: 2ml; sample flow rate: 4ml / min; experimental duration: 40min.

[0050] According to the order of sample peaks, the samples were divided into 6 fragments, and the female sample preparation fragments were named Preparationfragment of female 1-6 (PFF1-PFF6) ( Figure 2 A).

[0051] The different separated fragments were frozen and stored in a -80℃ refrigerator until freeze-dried. A freeze dryer was used to remove the methanol in the sample. During the drying process, boiling occurred, and the end of boiling indicated that the methanol was basically completely removed. The samples were stored in an ultra-low temperature refrigerator until the behavioral Y-maze experiment.

[0052] Repeat the above operation to collect different preparation fragments of 5-10 kDa samples of male Procambarus clarkii and name them as Preparation fragment of male 1-6 (PFM1-PFM6) ( Figure 2 B), served as the control group for subsequent metabolomics studies.

[0053] Behavioral results showed that among the six prepared fragments of 5-10 kDa obtained from female metabolic water, only fragment 6 (PFF6) could significantly attract male Procambarus clarkii individuals (χ 2 =18.233, P < 0.01), of which the experimental group was 114 and the control group was 58. At the same time, the remaining 5 isolated fragments (PFF1-PFF5) had no significant attraction effect on male Procambarus clarkii (P > 0.05) (Table 3). Therefore, female Procambarus clarkii fragment 6 (PFF6) was selected for subsequent metabolomics analysis, and male Procambarus clarkii fragment 6 (PFM6) was used as the control group for comparative screening of differential metabolites.

[0054] Table 3 Chi-square test table of behavioral experiments of male Procambarus clarkii on different prepared fragments

[0055] Prepared fragment name Number of control group selections Experimental group selection number No response <![CDATA[ 2 ]]> df P PFF1 92 89 19 0.05 1 0.824 PFF2 80 78 42 0.025 1 0.874 PFF3 80 72 48 0.421 1 0.516 PFF4 76 92 32 1.524 1 0.217 PFF5 84 93 23 0.458 1 0.499 PFF6 58 114 28 18.233 1 ＜0.01**

[0056] Step 4: Determination of differential metabolite composition (Experiment 4)

[0057] 4.1 Sample extraction

[0058] The frozen samples (female PFF6 and male PFM6) were slowly thawed at 4°C, and pre-cooled methanol / acetonitrile / water solution (2:2:1, v / v) was added, vortexed, low-temperature ultrasonicated for 30 min, allowed to stand at -20°C for 10 min, centrifuged at 4°C (14000g / min, 20 min), and the supernatant was vacuum dried. For mass spectrometry analysis, 100 μL of acetonitrile aqueous solution (acetonitrile: water = 1:1, v / v) was added for re-dissolution, vortexed, centrifuged at 14000g at 4°C for 15 min, and the supernatant was filtered through a 0.22 μm filter membrane for sample analysis.

[0059] 4.2 UPLC-MS / MS acquisition conditions

[0060] 4.2.1 Chromatographic conditions

[0061] The samples were separated using an Agilent 1290 InfinityLC ultra-high performance liquid chromatography system (UHPLC) HILIC column; column temperature 25°C; flow rate 0.5 mL / min; injection volume 2 μL; mobile phase composition A: water + 25 mM ammonium acetate + 25 mM ammonia, B: acetonitrile;

[0062] 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.

[0063] 4.2.2Q-TOF mass spectrometry conditions

[0064] The AB Triple TOF 6600 mass spectrometer (AB SCIEX) was used to collect the primary and secondary spectra of the samples:

[0065] Ion source: electrospray ionization (ESI) positive and negative ion sources;

[0066] Ion source temperature: 600°C;

[0067] Atomizing gas auxiliary heating gas 1 (Gas1): 60;

[0068] Auxiliary heating gas 2 (Gas2): 60;

[0069] Curtain air (CUR): 30psi;

[0070] Spray voltage (ISVF): ±5500V (positive and negative modes);

[0071] Primary mass-to-charge ratio detection range: 60-1000Da;

[0072] Secondary product ion mass-to-charge ratio detection range: 25-1000Da;

[0073] First-stage mass spectrometry scanning accumulation time: 0.20 s / spectra;

[0074] The secondary mass spectrometry scanning accumulation time was 0.05 s / spectra; the secondary mass spectra were acquired using the data-dependent acquisition mode (IDA), and the peak intensity value screening mode was used;

[0075] Declustering voltage (DP): ±60V (positive and negative modes);

[0076] Collision energy: 35 ± 15 eV;

[0077] Dynamic exclusion isotope ion range: 4Da.

[0078] 4.3 Metabolite classification analysis of RF6 and RM6 fragments prepared from Procambarus clarkii

[0079] The results of ultra-high performance liquid chromatography-mass spectrometry analysis showed that a total of 471 metabolites were identified from the PFF6 and PFM6 fragments of male and female Procambarus clarkii in positive and negative ion modes ( Figure 3 ), including lipids (Lipids and Lipid-like molecules: 39.703%), undefined substances (Undefined: 15.074%), organic acids and their derivatives (Organic acids and derivatives: 12.951%), benzene ring compounds (Benzenoids: 8.493%), organic oxygen compounds (Organic oxygen compounds: 7.431%), benzimidazole compounds (Organoheterocyclic compounds: 6.582%), organic nitrogen compounds (Organic nitrogen compounds: 5.096%), etc.

[0080] 4.4 Screening and selection of differential metabolites

[0081] Non-targeted metabolome analysis was used to compare and screen the metabolites of the female semi-prepared fragment PFF6 samples, and the male semi-prepared isolated fragment PFM6 samples were used as the control group. Multivariate statistical analysis was performed, and strict orthogonal partial least squares discriminant analysis (OPLS-DA) was used to compare the metabolites of the female semi-prepared fragment PFF6 samples. The metabolites of the male semi-prepared isolated fragment PFM6 samples were used as the control group. The variable importance projection value VIP>1, P value<0.05 and |log2 FC ||>1 (FC: fold difference) was used as the screening criterion for significantly differentially expressed metabolites to screen metabolites with significantly differential expression.

[0082] The bar graphs are used to visually display the up- and down-regulation of the significantly different metabolites screened in the positive and negative ion modes between the groups ( Figure 4). A total of 18 significantly differential metabolites (DMs) were screened from the male and female groups, of which 15 were upregulated in the female group and 3 were downregulated. The positive ion mode results showed that a total of 11 DMs were upregulated in the female group, including indole-3-acetonitrile, dimethyl sulfoxide (DMSO), nicotine, cotinine, tetraethylene glycol, myosmine, creatine, creatinine, glutaraldehyde, 4-aminophenol and 2-hydroxypyridine, and 1 downregulated DM was trigonelline ( Figure 4 A); A total of 6 DMs were obtained in the negative ion mode. Four DMs were upregulated in the female experimental group, including 2,4-dinitrophenol, Mestranol, Cis, cis-muconicacid, and Zinniol. The downregulated DMs included Tetradecylphosphonate and Trans-traumatic acid ( Figure 4 B).

[0083] Step 5: Determine the behavior of candidate information objects

[0084] Six endogenous and harmless DMs were screened out from the significantly differential metabolites DMs upregulated in Experiment 4, including anhydrous creatine, creatinine, cis-hexanedioic acid, 2-hydroxypyridine, mestranol, and indole-3-acetonitrile for behavioral experiments. The six differential metabolites were purchased from McLean Biochemical Technology Company and configured to a concentration of 0.01 mol / L. Mestranol and indole-3-acetonitrile are water-insoluble substances, and dimethyl sulfoxide (DMSO) was selected as the solvent. The control group of subsequent behavioral experiments was set to DMSO instead of ultrapure water. After the effective attractants were screened out, different concentrations were configured to determine their effective attraction concentration range for Procambarus clarkii. The concentration configuration range was 10 -7 mol / L-10 -3 mol / L. The behavioral experimental procedures and subsequent data processing methods refer to Experiment 1.

[0085] The results showed that among the six DMs selected, only mestranol could significantly attract male Procambarus clarkii individuals (χ 2＝10.215, P<0.01), but failed to attract female individuals significantly; the remaining five DMs had no significant sexual attraction to male and female Procambarus clarkii individuals (Table 4).

[0086] Mestranol CAS number: 72-33-3, molecular weight: 310.43, Chinese alias: 3-methoxy-17α-ethinyl estradiol, 17Α-ethinyl estradiol 3-methyl ether, ethinyl estradiol methyl ether, etc.

[0087] The results of the attraction of different concentrations of mestranol to male Procambarus clarkii showed that the lowest effective concentration of mestranol was 10 -6 mol / L-10 -5 mol / L, concentration ≥10 -5 mol / L, it can stably and significantly attract male Procambarus clarkii, and its attraction to male individuals increases with the increase of concentration ( Figure 5 ).

[0088] The mestranol obtained by screening in this experiment can be used to confuse or lure male Procambarus clarkii. During ecological control, the purpose of confusing male Procambarus clarkii can be achieved by releasing the new sex pheromone mestranol, thereby preventing male Procambarus clarkii from looking for female Procambarus clarkii for mating. In the breeding season, the new sex pheromone mestranol can also be used as an attractant to trap male Procambarus clarkii, achieving the purpose of ecological control. Mestranol can also be used 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 mestranol, extend the lure time of mestranol, and so on.

[0089] Table 4 Chi-square test of the effects of six DMs on the behavioral responses of male and female Procambarus clarkii

[0090]

[0091] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A new sex pheromone mestranol for use in confusing or luring male Procambarus clarkii, characterized in that: The concentration of mestranol is 10 -5 Up to 10 -3 mol / L.

2. The use of the novel sex pheromone mestranol as an active ingredient in the preparation of an attractant according to claim 1, characterized in that: The concentration of mestranol is 10 -5 Up to 10 -3 mol / L.

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