A kind of Chinese mitten crab pheromone and its application

Abstract

The present invention provides a pheromone of Chinese mitten crab, which can be conveniently attracted and trapped by applying carotenoids, avoiding biological invasion and maintaining ecological balance. On the other hand, during artificial seedling raising, the concentration of carotenoids in the water area can be detected, and by applying carotenoids, sexual aggregation and mating can be promoted, thereby improving the efficiency of artificial seedling raising.

Description

Technical Field

[0001] The invention relates to the field of agricultural chemistry, and in particular to a Chinese mitten crab pheromone and an application thereof. Background Art

[0002] Currently, the management and control of aquatic invasive species lags significantly behind that of terrestrial invasive species. Therefore, relevant agencies have been seeking an effective population management strategy to maintain low population levels for a long time. Based on the experience of existing aquatic animal management, species-specific chemical bait trapping methods are more beneficial to the ecological environment than traditional chemical killing and physical elimination methods. However, due to the lack of understanding of the molecular structure and release pattern of chemicals that regulate their life history and biological relationships, semiochemicals have not been widely used to control the population of aquatic invasive species.

[0003] The Chinese mitten crab is adapted to a variety of semi-terrestrial, freshwater and marine habitats and has spread around the world through ship ballast water discharge, escape from seafood markets and aquarium pet trade. By preying on invasive organisms, the Chinese mitten crab has caused serious ecological damage and changed the community structure of benthic invertebrates in freshwater and saltwater in the invaded areas. The Chinese mitten crab is a burrowing and combative nature, which is beneficial to its own survival, reproduction, population growth and habitat expansion. However, burrowing behavior increases erosion of levees, causes riverbank instability, and seriously hinders fishery management; competitive fighting behavior seriously inhibits the growth of local populations, gradually causing population homogeneity, destroying biodiversity and causing the ecosystem to collapse. The Chinese mitten crab has caused serious economic impacts. Due to its unique life history, the Chinese mitten crab is one of the rare invasive species that affects both freshwater and marine habitats. Therefore, it is urgent to develop technologies and methods to manage and control the number of Chinese mitten crabs.

[0004] Chinese mitten crab is an economic crab in China. In recent years, with the development of crab farming, crab seedlings have been in short supply and the scale of artificial seedling breeding has continued to expand. How to promote mating between male and female crabs and improve seedling breeding efficiency and yield are also urgent issues that need to be addressed. Summary of the invention

[0005] At present, the main method of prevention and control of Chinese mitten crabs is to capture and eliminate them manually. Due to the special living environment of Chinese mitten crabs and their habit of digging holes, the labor cost of manual capture is very high. If chemical elimination methods are used, it will cause indiscriminate elimination of organisms living in aquatic environments such as coastal zones and rivers, causing further deterioration of the ecosystem and may leave great ecological risks for future generations. Therefore, we start from the life history strategies and behavioral characteristics of Chinese mitten crabs to seek efficient and environmentally friendly population control technologies.

[0006] The Chinese mitten crab has a catadromous life cycle, including a freshwater adult stage, a saltwater reproductive stage, and a marine larval stage. In late autumn, sexually mature adults in streams and lakes migrate to the sea. Adults waiting to mate gather at estuaries to mate and lay eggs under the stimulation of salt water. Before mating, pairs of crabs undergo a brief period of courtship display and fighting before deciding to stay or separate. Unlike many crustacean species with a post-molting mating pattern, the female Chinese mitten crab is in a hard shell stage during mating, and the vulva cover undergoes decalcification in response to male courtship and indicates the female's receptivity. During the fighting stage of the mating process, females signal their "readiness" for mating by releasing a pheromone. This semiochemical is derived from the female's urine or vulval secretions. Therefore, we isolated and identified a sexual aggregation pheromone released by females during the mating stage. This active substance can attract male crabs to aggregate at a certain concentration, so reproductive separation and chemical sterilization can be achieved by manipulating pheromones.

[0007] At the same time, during the period suitable for mating between males and females, when the concentration of sex pheromones does not reach the aggregation concentration, sex pheromones can be artificially added to achieve the purpose of promoting mating.

[0008] The present invention provides a Chinese mitten crab pheromone, which is characterized by comprising daucosterol.

[0009] Preferred pheromones are sex pheromones or sex aggregation pheromones.

[0010] The present invention also provides a method for attracting, trapping, and / or gathering Chinese mitten crabs, characterized in that daucosterol is applied in water areas.

[0011] Preferably, daucosterol is applied to the water body so that the concentration of daucosterol is not less than 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, or 1.0 μg / mL.

[0012] Preferably, the Chinese mitten crab is a male crab.

[0013] The invention provides a method for promoting the mating of Chinese mitten crabs, which is characterized by applying daucosterol in water areas.

[0014] Preferably, daucosterol is applied to the water body so that the concentration of daucosterol is not less than 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, or 1.0 μg / mL.

[0015] The above method can also be used for preparing products, preparing reagents, preparing agricultural chemicals, preparing medicines, preparing pesticides, or preparing pheromones.

[0016] The invention also provides the use of daucosterol as the pheromone of Chinese mitten crab.

[0017] The present invention also provides an extract containing pheromone of Chinese mitten crab, which contains daucosterol.

[0018] The present invention also provides a method for preparing Chinese mitten crab pheromone, which is characterized by: collecting breeding water of sexually mature Chinese mitten crab females and males, and adsorbing them with macroporous resin; after the macroporous resin is eluted with ethanol, collecting the eluate, and concentrating to obtain an extract.

[0019] Preferably, the pheromone as described above is a sex pheromone or a sex aggregation pheromone.

[0020] Preferably, the macroporous resin is XAD-HP.

[0021] Preferably, the extract is further separated by solvent extraction, preferably into a petroleum ether phase, an ethyl acetate phase, an n-butanol phase and an aqueous phase.

[0022] Preferably, the ethyl acetate phase is separated, and the Chinese mitten crab pheromone is obtained by gel chromatography column chromatography with a dichloromethane-methanol ratio of 1:1 and silica gel column chromatography with a dichloromethane-methanol ratio of 20:1 to 5:1.

[0023] Preferably, the gel chromatography column is Sephadex LH-20.

[0024] Preferably, the Chinese mitten crab pheromone can be prepared into a preparation or composition together with a carrier or an auxiliary material.

[0025] Preferably, the preparation can be powder, tablet, sustained-release agent, controlled-release agent, aqueous solution, solution, emulsion, injection, etc.

[0026] The daucosterol of the present invention is a naturally occurring substance, which is non-toxic and harmless to the environment. On the one hand, by applying daucosterol, it is convenient to attract and trap; it can make Chinese mitten crabs gather in a specific area, achieve reproductive separation, chemical sterilization or harmless treatment, avoid biological invasion, and maintain ecological balance; at the same time, it can save a lot of manpower and material resources required for artificial capture, and avoid the use of toxic and harmful substances. On the other hand, for the aquaculture industry, during the artificial seedling raising period, the concentration of daucosterol in the water area can be detected. When the concentration of daucosterol in the water area is lower than the concentration required for sexual aggregation, daucosterol can be applied to promote sexual aggregation and mating, thereby improving the efficiency of artificial seedling raising. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : The chemical structural formula of daucosterol.

[0028] Figure 2 :The content of daucosterol in water bodies, A) Compound release (ng / mL), SM, male crab after seawater stimulation; SF, female crab after seawater stimulation; SMF, male and female crabs mating after seawater stimulation; ***P<0.01B) The content of daucosterol in the hemolymph of female crabs changes with the lunar phase. November 8, 2022 is a full moon.

[0029] Figure 3 : Test model for the Chinese mitten crab aggregation assay.

[0030] Figure 4 : The cumulative time (%) of the tested Chinese mitten crab in the test waterway, the test samples were A) seawater vs. seawater; B) female crab culture water vs. seawater; C) mating culture water vs. seawater; D) daucosterol vs. seawater. Each test sample was tested three times in the waterway, ***P<0.01

[0031] Figure 5 : Heat map of Chinese mitten crab movement, A) blank, both arms were filled with seawater; B) test, the left arm was treated with daucosterol, and the right arm was filled with seawater. The degree of movement aggregation was qualitatively determined by the colorimetric bar. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the embodiments.

[0033] Example 1: Isolation and purification of Chinese mitten crab sexual aggregation pheromone

[0034] About 60 sexually mature female crabs (weighing about 100 grams) and 30 sexually mature male crabs (weighing about 150 grams) were raised in a seawater environment. The culture water was collected every day and adsorbed using a macroporous resin (XAD-HP, 2 kg) for a total of 60 days. After the macroporous resin was eluted with ethanol, about 27.2 grams of crude extract was collected by vacuum distillation, and after further solvent extraction and phase separation, an ethyl acetate phase (about 8.6 grams) was obtained. The organic phase was further chromatographically separated to obtain 19 components, and component 12 (about 1.1 grams) was further purified, eluted by gel column chromatography (dichloromethane-methanol 1:1), and after silica gel column chromatography (dichloromethane-methanol 20:1 to 5:1), a monomer compound (5.1 mg) was obtained.

[0035] Example 2: Chemical structure of the sexual aggregation pheromone of Eriocheir sinensis

[0036] The compound was identified as daucosterol by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy.

[0037] Daucosterol: amorphous powder; 1 H and 13 C NMR data, Table 1; HRESIMS m / z 575.4321[M–H] – (calcd for C 35 H 59 O6,575.4312,Δ=1.6ppm). Its chemical structure is as follows Figure 1 shown.

[0038] Table 1. NMR spectra of compounds (DMSO, 1 H at 500MHz, 13 Cat at 125MHz)

[0039]

[0040] Example 3: Implementation dosage of Chinese mitten crab sexual aggregation pheromone

[0041] The quantitative analysis method of daucosterol in biological media was developed and verified by ultra-high performance liquid chromatography-mass spectrometry (Ultimate3000-TSQ Quantiva). The chromatographic conditions of this method are as follows: analytical column: HypersilGOLD column (50×2.1mm, 1.9μm); mobile phase: phase A: 1mM ammonium acetate aqueous solution; phase B: methanol (containing 1mM ammonium acetate). Elution conditions: 0.0-2.0min, 10-95% B; 2.0-6.0min, 95% B; 6.0-6.1min, 95-10% B; 6.1-7.5min, 10% B; injection volume: 1μL; flow rate: 0.3mL / min, column temperature: 35℃. The mass spectrometry conditions of this method are as follows: the ion source is an electrospray ionization source (ESI); the ionization voltage is 4500V; the source temperature is 500°C; both GS1 and GS2 are 50psi; negative ion detection; the collision energy, declustering voltage and ion reaction used for quantitative analysis are shown in Table 2.

[0042] Table 2 Mass spectrometry conditions for quantitative analysis of daucosterol

[0043]

[0044] This method was used to analyze the male and female crabs stimulated by seawater, and the water bodies where male and female crabs were mixed ( Figure 2 ), and found that during mating, the daucosterol content reached about 848.8ng / mL, from which we speculated that this dose had an aggregation effect. We then measured the peritoneal fluid of female and male crabs separately and found that the compound came from female crabs waiting to mate ( Figure 2 ).

[0045] Example 4: Aggregation effect test

[0046] In the Y-shaped maze ( Figure 3 ), we tested the aggregation effect of the samples. First, seawater was added to the left and right arms to verify that the crabs had no directional preference. Then, a seawater solution containing daucosterol was added to one arm at a flow rate of about 200 mL / min. The movement trajectory of the test animals was recorded with a camera.

[0047] The model was used to count the cumulative time that the test animals spent in the test waterway, and the results were as follows: Figure 4 shown. Figure 4 A indicates that the Chinese mitten crabs have no preference for the direction of the waterway. Figure 4 B indicates that the breeding water of female crabs is not attractive to male crabs. Figure 4 C indicates that the culture water during mating has an attractive effect on male crabs (65.38±9.02%). Figure 4 D shows that the seawater solution of daucosterol (1.0 μg / mL, flow rate 200 mL / min) has an attractive effect on male crabs (66.80±5.33%), which is significantly different from blank seawater and can be repeated to show the cumulative percentage of attraction time of the culture water during mating.

[0048] Figure 5 A means that Chinese mitten crab has no preference for the direction of the waterway. Figure 5 B indicates a preference for the stimulus solution containing daucosterol on the left.

[0049] It can be seen from the experiment of the present invention that daucosterol, as a necessary sexual aggregation pheromone for the mating process of male and female Chinese mitten crabs, plays an extremely important role in the mating process. Accordingly, daucosterol has two applications. One is that it can be used for attraction and trapping, so that Chinese mitten crabs can be gathered in a specific area, reproductive separation, chemical sterilization or harmless treatment can be achieved, biological invasion can be avoided, and ecological balance can be maintained; at the same time, a large amount of manpower and material resources required for artificial capture can be saved, and toxic and harmful substances can be avoided. Second, for the aquaculture industry, during the artificial seedling raising period, the concentration of daucosterol in the water area can be detected. When the concentration of daucosterol in the water area is lower than the concentration required for sexual aggregation, daucosterol can be applied to promote sexual aggregation and mating, thereby improving the efficiency of artificial seedling raising.

[0050] The above description is a general description of the present invention. According to the situation or actual needs, changes in form and substitution of equivalents can be made. Although specific terms are used herein, these terms are intended to describe rather than limit the purpose. Those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for attracting, trapping, and / or gathering Chinese mitten crabs, characterized in that Daucosterol is applied in the water area, wherein the concentration of the daucosterol is not less than 1.0 μg / mL, and the Chinese mitten crab is a male crab.

2. Use of daucosterol as pheromone of Chinese mitten crab.

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