A biological agent for controlling mosquito larvae

Abstract

The present invention discloses a biological preparation for preventing and controlling mosquito larvae, wherein the active ingredients are composed of six toxic proteins, namely Cry54Ba, Cry50Ba1, Tpp80Aa1, Mpp82Aa1, Xpp88Aa1 and Cyt10Aa, and the synergistic effect is most significant when the molar ratio of the six toxic proteins is 1:1. After mixing in equal molar ratios, a higher mosquito killing activity is produced through the synergistic action of the toxic proteins, and the mosquito killing rate reaches 100%. Therefore, the combination preparation of multiple toxic proteins produces a highly effective killing activity on the harassing mosquito larvae, effectively reduces the reproduction of mosquitoes, and has a significant effect on the prevention and control of mosquitoes, and the mosquito killing toxic protein preparation of microbial origin has the characteristics of high efficiency and specificity, green environmental protection, and safety and friendliness to non-target organisms.

Description

Technical Field

[0001] The invention belongs to the technical field of pesticides, and in particular relates to a biological preparation for preventing and controlling mosquito larvae. Background Art

[0002] Mosquitoes are insects of the Culicidae family in the order Diptera. There are more than 3,600 species of mosquitoes recorded worldwide, mainly belonging to three subfamilies (Anopheles, Culex, and Gastrofasciatinae) and 43 genera. The genus Culex is the vector of viral encephalitis and spreads filariasis in tropical and subtropical areas. Different species of mosquitoes have similar living habits. Among them, the harassing mosquito (Armigeres subalbatus) belongs to the subfamily Culex, which mainly sucks blood and causes harassment. When it bites and sucks blood, it is extremely itchy and painful; in addition, it is also a vector of mosquito-borne infectious diseases (such as filariasis, viral encephalitis, etc.) and Zika virus, which has attracted the attention of workers in the fields of biomedicine and health and epidemic prevention.

[0003] The larvae of the harassing mosquito breed in puddles of varying sizes or depths or in manure pits. The population density is high, and they spend the winter in larval form. Adults mostly live in rooms, animal sheds, and toilets. They increase their blood-sucking activities at dusk and dawn, and can also suck blood during the day when environmental conditions are suitable. With the rapid development of productivity, modern high-rise buildings in human living space have raised the starting point of mosquito breeding space, thereby expanding their living area. As a result, even people living far away from shallow waters are troubled by harassing mosquitoes.

[0004] At present, the main strategies for mosquito control include four aspects: Environmental control: mainly by improving the habitat environment and people's living conditions and habits, so as to reduce the places where mosquitoes breed, thereby reducing the source of mosquitoes. By changing the conventional flooding in the river basin area and promoting wet irrigation of rice fields, the mosquito breeding grounds can be changed to effectively control the number of mosquitoes. Usually, this non-toxic mosquito control measure is suitable for nature reserves. Physical control: using physical means or physical equipment to disinfect adult mosquitoes, such as screen doors and windows, mosquito nets, electric mosquito swatters, ultrasonic mosquito killing equipment, physical mosquito killing devices, etc. Chemical control: mainly using chemical pesticides to disinfect adult mosquitoes. Indoor spraying of DDT, malathion, pyrethroids and other mosquito killers is mainly used to control mosquitoes. Although chemical methods have the advantages of high efficiency and quick effect in controlling mosquitoes, they pollute the environment, have high residues, and even affect human health. Biological control: directly using the organism itself or biological metabolites to prevent and control another organism. Its advantages are: biological mosquito killers are safe to operate and do not pollute the environment. The biological insecticide Bt has a high efficiency in killing mosquitoes and has become a global research hotspot. Under the background of today's green development concept, it is urgent to develop new agents that are efficient, green, non-toxic and long-lasting for the prevention and control of mosquito larvae. Summary of the invention

[0005] Due to the selection pressure of long-term high-intensity and continuous use, many insects have developed resistance to existing Bt insecticide products. The purpose of the present invention is to provide a biological agent for controlling mosquito larvae, so as to improve the control effect of mosquito larvae, delay or overcome the drug resistance of mosquitoes, reduce the use of chemical pesticides, and reduce pollution to the environment.

[0006] To achieve the above objectives, the present invention provides a biological preparation for controlling mosquito larvae, wherein the active ingredients are composed of six toxic proteins: Cry54Ba, Cry50Ba1, Tpp80Aa1, Mpp82Aa1, Xpp88Aa1 and Cyt10Aa.

[0007] Furthermore, the molar ratio of the six toxic proteins is 1:1:1:1:1:1:1 (ie, the molar ratio of the six toxic proteins is 1:1).

[0008] Furthermore, the mosquito is an Aedes aegypti.

[0009] Furthermore, the active ingredient is 1 to 5% by weight of the biological preparation.

[0010] Furthermore, the active ingredient accounts for 2-3% of the weight percentage of the biological preparation.

[0011] Furthermore, the active ingredient is 3% by weight of the biological preparation.

[0012] Furthermore, the biological agent for controlling mosquito larvae also includes auxiliary materials, which are composed of sodium alginate solution, calcium chloride solution, hollow glass microspheres, melanin, sodium benzoate solution, fish meal and water.

[0013] The biological agent for preventing and controlling mosquito larvae is used in preventing and controlling larvae of Aedes aegypti, wherein the mosquito larvae are larvae of Aedes aegypti.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects:

[0015] 1. The combination of Cry54Ba, Cry50Ba1, Tpp80Aa1, Mpp82Aa1, Xpp88Aa1 and Cyt10Aa has a significant synergistic effect, among which the synergistic effect is most significant when multiple toxic proteins are mixed in equal molar ratios. 50The single toxic protein in the combination has a certain insecticidal activity against mosquito larvae. The equal proportion of the toxic proteins in the combination produces a higher mosquito killing activity through synergistic effect, and the mosquito killing rate is as high as 100%. Therefore, the combination of multiple toxic proteins has a high efficiency in killing mosquito larvae, effectively reducing the reproduction of mosquitoes and playing a significant role in the prevention and control of mosquitoes. Microbial-derived mosquito-killing toxic protein preparations are characterized by high efficiency, specificity, green environmental protection, and safety and friendliness to non-target organisms.

[0016] 2. The present invention relates to a combination of multiple toxic proteins, which can delay or overcome the emergence of mosquito resistance and extend the service life of the preparation.

[0017] 3. The present invention has a good control effect on the harassing mosquito, and the control effect on the harassing mosquito is as high as 100% within 24 hours after the drug is applied. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] The toxic proteins in the present invention are all existing proteins, from Zhou Yan, De Novo sequencing and mass spectrometry proteome analysis revealed that the Bt S2160-1 strain may have a mosquito-killing toxic protein island, Guangxi University doctoral dissertation, 2020, among which Tpp80Aa1 corresponds to Cry80Aa1 in this doctoral dissertation, Mpp82Aa1 corresponds to Epp in this doctoral dissertation, Xpp88Aa1 corresponds to the 2118 gene in this doctoral dissertation (S3580-1CG_2118), and Cyt10Aa corresponds to the 237 gene in Chapter 5 of this doctoral dissertation (pS2160-1p2_gene_id_237).

[0024] The total protein of mosquito-killing strain Bt S2160-1 (Bacillus thuringiensis S2160-1) was purchased from the General Microbiology Center of China Microbiological Culture Collection Administration.

[0025] Example 1

[0026] Determination of the biological activity of toxic protein against Argyi mosquitoes

[0027] The bioassay of mosquitoes was carried out according to the standard procedure established by WHO. The total volume of all bioactivity assays was controlled to be 5 mL. The six protein solutions of Cry54Ba, Cry50Ba1, Tpp80Aa1, Mpp82Aa1, Xpp88Aa1, and Cyt10Aa with calibrated concentrations were diluted into 5 concentration gradients (50 μg / mL, 150 μg / mL, 250 μg / mL, 350 μg / mL, and 500 μg / mL) with dechlorinated tap water, respectively, and placed in a single well of a sterilized 6-well plate, and the volume was filled to 5 mL with distilled water, and the mixture was stirred thoroughly. Thirty 3rd-instar mosquito larvae were transferred into the plate using a Pasteur tube. Dechlorinated tap water without any protein was set as a negative control, and the total protein of the mosquito-killing strain Bt S2160-1 was set as a positive control. Three replicates were set for each protein concentration. All bioassays were conducted in an artificial constant environment with a temperature of (27±1)℃, a relative humidity of (65±5)%, and a photoperiod of 14L:10D. The number of dead and alive mosquito larvae was observed and recorded every day. After 3 days, the data were counted and the toxicity regression analysis was performed using SPSS analysis software to calculate the LC 50 value.

[0028] In addition, each combination including Cry54Ba+Cry50Ba1, Cry54Ba+Cry50Ba1+Tpp80Aa1, Cry54Ba+Cry50Ba1+Tpp80Aa1+Mpp82Aa1, Cry54Ba+Cry50Ba1+Tpp80Aa1+Mpp82Aa1+Xpp88Aa1 and Cry54Ba+Cry50Ba1+Tpp80Aa1+Mpp82Aa1+Xpp88Aa1+Cy t10Aa was tested for biological activity at an equimolar ratio. Other experimental methods were performed according to single protein.

[0029] The results are shown in Table 1.

[0030] Table 1 Mosquito-killing activity test results

[0031]

[0032] From the results in Table 1, it can be seen that the single toxic proteins in the combination have certain insecticidal activity against mosquito larvae, but not all toxic proteins have synergistic effects after combination. The combination of Cry54Ba, Cry50Ba1, Tpp80Aa1, Mpp82Aa1, Xpp88Aa1 and Cyt10Aa has significant synergistic effect, among which the synergistic effect is most significant when the six toxic proteins are mixed in equal molar ratios, and their LC 50 It is 843.5ng / mL.

[0033] Example 2

[0034] A biological agent for preventing and controlling larvae of Argyi mosquitoes, wherein the active ingredients are composed of six toxic proteins, namely Cry54Ba, Cry50Ba1, Tpp80Aa1, Mpp82Aa1, Xpp88Aa1 and Cyt10Aa, wherein the molar ratio of the six toxic proteins is 1:1, and the auxiliary materials are composed of sodium alginate solution, calcium chloride solution, hollow glass microspheres, melanin (water-soluble), sodium benzoate solution and fish meal. The weight percentages of various raw materials in the biological agent are as follows:

[0035] raw material

[0036]

[0037] A method for preparing a biological agent for preventing and controlling harassing mosquito larvae is prepared by the following steps:

[0038] (1) accurately weighing the raw materials according to the above weight percentages, mixing sodium alginate, six toxic proteins, raw powder, melanin (water-soluble), sodium benzoate, hollow glass microspheres, fish meal and water in equal molar ratios to prepare a mixed solution;

[0039] (2) preparing a calcium chloride solution having a weight percentage concentration of 3.5%;

[0040] (3) using a 10 mL sterile syringe, the mixed solution of step (1) is added dropwise to the calcium chloride solution of step (2), and the droplets are formed into particles and solidified;

[0041] (4) The solidified droplets of step (3) were hardened in a calcium chloride solution for 4 h. The calcium chloride solution was discarded, and the hardened granular microbeads were washed twice with distilled water, air-dried, and then stored at room temperature.

[0042] Example 3

[0043] Same as Example 2, except that the weight percentages of various raw materials in the biological preparation are as follows:

[0044] raw material

[0045]

[0046] Example 4

[0047] Same as Example 2, except that the weight percentages of various raw materials in the biological preparation are as follows:

[0048] raw material

[0049]

[0050]

[0051] Example 5

[0052] Same as Example 2, except that the weight percentages of various raw materials in the biological preparation are as follows:

[0053] raw material

[0054]

[0055] Weigh 1g of the biological agent for killing the larvae of Aedes aegypti of Example 2-5 of the present invention and add it to 10L of water, put in 100 3rd instar larvae of Aedes aegypti, repeat the experiment 3 times, and use water as a negative control. Observe the 24h mortality rate. When the treatment mortality rate exceeds 50%, replace with fresh water and move in new test larvae; replace fresh water and fresh test larvae uniformly every 24h. The effects of Example 2-5 of the present invention are shown in Table 2, and the suspension rate is shown in Table 3.

[0056] Table 2 Corrected mortality of 3rd instar larvae of Aedes mosquitoes harassed at different times after treatment with biological agents that kill mosquito larvae

[0057]

[0058]

[0059] Note: The above data results are the average of 3 repetitions.

[0060] Table 3 Average suspension rate at different times after treatment to kill harassing mosquito larvae

[0061]

[0062] The results in Table 2 show that after using different contents of biological agents to treat the larvae of the mosquito, the mortality rate reached 100% after 72 hours; the mosquito killing effect of Examples 4 and 5 is better than that of Examples 2 and 3. In addition, it can be seen from Table 3 that the suspension of the biological agent for controlling the larvae of the mosquito is long-lasting, and it began to sink after 168 hours, and the suspension rate was still 65.2% at 216 hours. Compared with Examples 4 and 5, when the durability and insecticidal effect are almost the same, Example 4 is more suitable for application and production, that is, the original powder mixed with six toxic proteins at a lower content can achieve the same mosquito killing effect.

[0063] The above are only preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A biological agent for controlling mosquito larvae, characterized in that: The active ingredients are composed of six toxic proteins: Cry54Ba, Cry50Ba1, Tpp80Aa1, Mpp82Aa1, Xpp88Aa1, and Cyt10Aa; The molar ratio of the six toxic proteins is 1:1:1:1:1:1:

1.

2. A biological agent for controlling mosquito larvae according to claim 1, characterized in that: The mosquito is Aedes aegypti.

3. A biological agent for controlling mosquito larvae according to claim 1, characterized in that: The active ingredient accounts for 1 to 5% of the weight percentage of the biological preparation.

4. A biological agent for controlling mosquito larvae according to claim 3, characterized in that: The active ingredient accounts for 2-3% of the weight percentage of the biological preparation.

5. A biological agent for controlling mosquito larvae according to claim 4, characterized in that: The active ingredient is 3% by weight of the biological preparation.

6. Use of the biological agent for controlling mosquito larvae according to any one of claims 1 to 5 in controlling Argonaut larvae.

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