Insect-free medium for artificial culture of trichogramma and preparation method and application thereof

Abstract

The present application belongs to the field of artificial breeding of natural enemy insects, and particularly relates to a Trichogramma artificial culture medium without insect medium and a preparation method and application thereof. The Trichogramma artificial culture medium comprises chicken egg yolk, TNM-FH culture medium, Nissl salt solution, trehalose solution and anhydrous lactose solution; further comprises an amino acid mixture, B group vitamins and yeast extract. The artificial culture medium without insect medium can be used for in vitro culture in the process of artificial breeding of Trichogramma, and the artificial culture medium system capable of supporting the continuous development of Trichogramma is constructed under the premise of not using insect source medium, and is suitable for the continuous development support of Trichogramma from the egg stage to the adult emergence stage.

Description

Technical Field

[0001] This invention belongs to the field of artificial breeding of natural enemy insects, specifically relating to an artificial culture medium for Trichogramma wasps without insect media, its preparation method, and its application. Background Technology

[0002] Red-eyed bee ( Trichogramma (This is a fragment of a sentence and doesn't translate directly.) It is one of the most widely used parasitic wasp groups in the biological control of agricultural pests, and its large-scale breeding usually relies on rice moths (This is a fragment of a sentence and doesn't translate directly.) Corcyra cephalonica ), oak silkworm ( Antheraea pernyi (The text then abruptly shifts to discussing the use of insects as host eggs or substitute host eggs. While existing artificial culture techniques have reached a certain level, most still rely on insect-derived substances such as insect hemolymph and insect tissue extracts as key culture components, and a stable, reproducible insect-free culture system has not yet been established. Previous studies have shown that artificial culture can support the early development of Trichogramma wasps to some extent, but there are still significant shortcomings in terms of pupation rate, emergence rate, and adult wasp quality.)

[0003] The main problems with existing technologies are: first, they are highly dependent on insect-derived raw materials, resulting in high raw material acquisition costs and large batch-to-batch variations, which is not conducive to standardized production; second, the nutritional composition of the culture medium and key developmental regulatory factors are not yet clear, making it difficult to simultaneously meet the needs of different developmental stages of Trichogramma wasps. The fundamental reason is that existing technologies still lack sufficient understanding of the core nutrients and promoting factors required for the in vitro development of Trichogramma wasps, thus limiting the establishment and application of insect-free artificial culture media.

[0004] Therefore, it is necessary to address the problems of existing artificial culture techniques for Trichogramma wasps, such as strong dependence on insect-derived components, unclear culture medium composition, insufficient stability and reproducibility, and difficulty in effectively supporting the continuous development of Trichogramma wasps. This paper proposes an insect-free artificial culture medium for Trichogramma wasps and its preparation method. This culture medium can meet the nutritional requirements of Trichogramma wasps for in vitro development, improve their survival rate, pupation rate, and emergence rate during artificial culture, thereby providing technical support for the standardized and large-scale breeding of Trichogramma wasps. Summary of the Invention

[0005] This invention provides an insect-free artificial culture medium for Trichogramma wasps, as well as a method for preparing and applying the medium. The technical solution constructs a basic culture system using non-insect-derived nutrient components, and then optimizes and supplements this system with key active substances that promote different developmental stages of Trichogramma wasps, thereby forming an artificial culture medium system capable of supporting the continuous growth of Trichogramma wasps from the egg stage to subsequent developmental stages.

[0006] The technical solution of this invention is as follows: The first objective of this invention is to provide an insect-free artificial culture medium for Trichogramma wasps, comprising a basal culture medium including: egg yolk, TNM-FH medium, Nissl salt solution, trehalose solution, and a penicillin-streptomycin mixture; the artificial culture medium for Trichogramma wasps also includes an amino acid mixture, B vitamins, and yeast extract.

[0007] Egg yolks, as a source of lipids, lipoproteins, and some nutrients, are used to maintain the needs of Trichogramma wasps for membrane structure formation and basic nutrient supply during early development. TNM-FH medium is used to provide a liquid culture environment and the necessary buffer system, which helps maintain the stability of the culture system; Nissl salt solution is used to regulate the ionic environment in order to maintain the electrolyte balance of the culture medium; Trehalose solution is used to provide available energy and help maintain osmotic pressure stability; Penicillin-streptomycin mixture is used to inhibit microbial contamination and improve the stability of the culture process.

[0008] Amino acids are used to improve the survival rate of Trichogramma wasps during the egg-larval stage; B vitamins are used to promote their development from the egg-larval stage to the prepupal stage; yeast extract is used to improve the subsequent pupation rate and emergence rate.

[0009] Furthermore, the volume ratio of egg yolk, TNM-FH insect cell culture medium, Nissl salt solution, 0.1 g / mL trehalose solution, and penicillin-streptomycin mixture (100X) (containing 10000 u / ml penicillin and 10000 μg / ml streptomycin) in the basal culture medium is 1:1:0.3:0.1:0.1.

[0010] Furthermore, the Nissl salt solution is prepared by adding 7.5g NaCl, 0.2g NaHCO3, 0.2g CaCl2, and 0.1g KCl to distilled water and bringing the volume to 100 mL.

[0011] Furthermore, the penicillin-streptomycin mixture contains 10,000 u / ml of penicillin and 10,000 μg / ml of streptomycin, and the penicillin-streptomycin mixture is added in equal volume to the trehalose solution.

[0012] Furthermore, after the amino acid mixture is prepared into an amino acid mixed solution, it is added to the artificial culture medium of Trichogramma bees. The concentrations of each amino acid in the amino acid mixed solution are as follows: Table 1. Amino acid mixture formulation β-alanine 238 L-Lysine hydrochloride 535 glycine 568 L-methionine 230 L-alanine 671 L-phenylalanine 117 L-arginine hydrochloride 476 L-proline 1385 L-Asparagine 0.9 L-serine 960 L-Cystine Dihydrochloride 2.4 L-threonine 868 L-glutamic acid 3761 L-Tryptophan 74 L-glutamine 2155 L-tyrosine disodium salt 384 L-histidine hydrochloride monohydrate 1071 L-valine 284 L-Isoleucine 425 ornithine 88 L-Leucine 330 Furthermore, in the artificial culture medium for Trichogramma bees, the volume ratio of the amino acid mixture solution to the basal culture medium is 1:25.

[0013] Furthermore, in the artificial culture medium for Trichogramma bees, B vitamins are prepared into a mixed solution and then added to the artificial culture medium. The concentrations of each B vitamin in the mixed solution are as follows: In the artificial culture medium for Trichogramma bees, B vitamins were prepared into a mixed B vitamin solution and then added to the culture medium. The concentrations of each B vitamin in the mixed B vitamin solution were as follows: thiamine (VB1) 703±10 ng / mL, riboflavin (VB2) 12528±10 ng / mL, niacin (VB3) 1455±10 ng / mL, nicotinamide (VPP) 165±10 ng / mL, pantothenic acid (VB5) 41546±10 ng / mL, pyridoxal (VB6) 0.27±0.03 ng / mL, pyridoxal 5-phosphate (PLP) 478±10 ng / mL, biotin (VB7) 647±10 ng / mL, and folic acid (VB9) 73±10 ng / mL. The concentration of cobalamin (VB12) was 0.27 ± 0.03 ng / mL.

[0014] In a specific embodiment, the concentrations of each B vitamin in the B vitamin mixed solution were as follows: thiamine (VB1) 703.8239214 ng / mL, riboflavin (VB2) 12528.56045 ng / mL, niacin (VB3) 1455.250656 ng / mL, nicotinamide (VPP) 165.5889118 ng / mL, pantothenic acid (VB5) 41546.43681 ng / mL, pyridoxal (VB6) 0.274839447 ng / mL, pyridoxal 5-phosphate (PLP) 478.2480984 ng / mL, biotin (VB7) 647.6303127 ng / mL, and folic acid (VB9) 73.69184619 ng / mL. The concentration of cobalamin (VB12) was 0.275097117 ng / mL.

[0015] Furthermore, in the artificial culture medium for Trichogramma bees, the volume ratio of the B vitamin mixed solution to the basal culture medium is 1:25.

[0016] Furthermore, the yeast extract is added after being prepared into a yeast extract solution with a concentration of 0.2 g / mL. In the artificial culture medium for Trichogramma bees, the volume ratio of the added yeast extract solution to the basal culture medium is (0.2-0.6):25.

[0017] A second objective of this invention is to provide the application of the aforementioned artificial culture medium for Trichogramma wasps in in vitro culture during the artificial breeding process of Trichogramma wasps.

[0018] The insect-free artificial culture medium described in this invention can be used for in vitro culture during the artificial breeding of Trichogramma wasps, and is especially suitable for continuous development support during the egg stage, larval stage, prepupal stage, pupal stage and even the adult emergence stage of Trichogramma wasps.

[0019] In application, the culture medium can be loaded into artificial eggshells or other suitable artificial carriers to simulate the internal nutrition and liquid environment of the host egg, providing food and larvae of Trichogramma wasps for feeding and development.

[0020] To ensure successful cultivation, female Trichogramma wasps that have emerged within 24 hours, have fully mated, and have not yet laid eggs should be selected as the inoculation source. Prepared egg cards should be placed one by one in a clean container for inoculation. The optimal wasp-to-egg ratio during inoculation is 8:1. Strong external light should be avoided during inoculation; 24 hours of shading can be used for cultivation.

[0021] In one embodiment of the present invention, the subsequent culture conditions can be set as follows: temperature 25±1℃, relative humidity 75%±5%, photoperiod 14L∶10D, light intensity 3 Lux; after receiving bees for 24 hours, remove the bees and then transfer the egg cards after receiving the bees to the dark conditions for continued culture, so as to observe the subsequent development, pupation and emergence of Trichogramma wasps.

[0022] In order to reduce the impact of environmental fluctuations on the quality and biological traits of Trichogramma wasps during the maintenance and synchronous management of insect sources, Trichogramma wasp populations can be routinely cultured and managed under the conditions of temperature 25±1℃, relative humidity 70%±5%, and photoperiod L14∶D10, so as to ensure that the generational background and physiological state of the tested individuals are as consistent as possible.

[0023] The beneficial effects of this invention are as follows: This invention constructs an artificial culture medium system that can support the continuous development of Trichogramma wasps without using insect-derived media. By supplementing key active substances at different developmental stages, it supports key developmental stages of Trichogramma wasps, such as egg-larval survival, prepupa formation, pupation, and emergence.

[0024] Compared with existing culture systems that rely on insect-derived raw materials such as insect hemolymph, the culture medium described in this invention has the advantages of relatively clear raw material sources, easier system standardization, easier batch-to-batch stability control, and greater room for cost optimization. Therefore, it is suitable for the further establishment and improvement of the artificial breeding system of Trichogramma wasps. Specifically: 1. To support the continuous development of Trichogramma wasps without using insect-derived media. This invention constructs an artificial culture system using non-insect-derived components, avoiding reliance on insect-derived raw materials such as insect hemolymph and insect tissue homogenates. This overcomes the technical limitations of existing culture systems, which suffer from high dependence on host-derived components, limited raw material availability, and significant batch-to-batch variations. This culture medium provides continuous support for the development of *Trichogramma* wasps from the egg stage, larval stage, prepupal stage, pupal stage to adult emergence, demonstrating that this invention not only achieves component substitution in the culture medium but also effectively inherits the developmental support function.

[0025] 2. It has a targeted ameliorative effect on key limiting factors at different developmental stages of Trichogramma wasps. This invention does not simply provide basic nutrient solutions, but rather optimizes the culture medium to meet the physiological needs of Trichogramma wasps at different developmental stages.

[0026] Among them, the amino acid mixture can support the survival of Trichogramma wasp eggs and larvae. A mixture of B vitamins can promote the development of Trichogramma wasps from the egg-larval stage to the prepupal stage. The results showed that supplementing the basal culture medium containing amino acids with B vitamins increased the prepupal rate from 55.96% to 83.46%, indicating that this component has a significant promoting effect on overcoming early developmental stagnation. Yeast extract significantly improved subsequent pupation and emergence. The results showed that after further supplementing yeast extract with the basal culture medium containing amino acids and vitamins, the pupation rate increased from 9.10% to 82.44%, and the emergence rate increased from 2.57% to 27.22%, with highly significant differences (p<0.0001; p<0.0001).

[0027] The above results demonstrate that the culture medium system established in this invention can effectively improve key developmental bottlenecks in the in vitro culture of Trichogramma wasps, and has clear experimental evidence.

[0028] 3. The composition of the culture medium raw materials is more clearly defined, which facilitates standardized preparation and quality control. The culture medium components used in this invention have clearly defined sources and a controllable preparation process, making it easier to standardize formulations, prepare in batches, and repeat processes compared to culture systems that rely on natural insect-derived materials. Since natural materials such as insect hemolymph typically exhibit individual differences, variations in the time of collection, and fluctuations in composition, the technical solution described in this invention, by clearly defining the basic culture system and functional supplementary components, helps improve the batch-to-batch stability and repeatability of the culture medium, thus providing a foundation for subsequent standardized production and quality control.

[0029] 4. It helps simplify the raw material acquisition process and improves the operability of applications. In existing technologies, if the culture system relies on host insect hemolymph or other raw materials, it is usually necessary to simultaneously maintain the host insect population, collect biological materials, and process them, resulting in numerous operational steps and making the stability of raw material supply easily limited. This invention, by employing an insect-free medium culture scheme, reduces direct reliance on live host insect materials, thus simplifying the raw material preparation process and improving the convenience and feasibility of the culture system.

[0030] 5. It provides a new technical approach for establishing an artificial breeding system for Trichogramma wasps. The significance of this invention lies not only in obtaining a culture medium formulation, but also in demonstrating that some key developmental support functions during the artificial culture of Trichogramma wasps can be replaced by non-insect-derived nutrient modules. This indicates that the key factors required for the in vitro culture of Trichogramma wasps do not necessarily have to be derived from natural insect hemolymph, but can be achieved through artificially constructed culture systems.

[0031] Therefore, this invention provides a new technical foundation and application direction for the subsequent artificial breeding, large-scale production, and optimization of related culture systems of Trichogramma wasps. Attached Figure Description

[0032] Figure 1 Amino acid testing, including: A: Number of Trichogramma wasp eggs per artificially created egg; B: Survival rate; The error bar in the figure represents the standard error (SEM). The values ​​in the figure are the mean, and the error bar represents the standard error. Independent samples t-tests were used for comparisons between groups, with ** indicating a highly significant p-value < 0.0001.

[0033] Figure 2 B vitamins activity test, including: A: Number of Trichogramma eggs per artificial egg; B: Prepupae rate; C: Pupation rate; The values ​​in the figure are the mean, and the error bars are the standard errors. Independent samples t-tests were used for comparisons between groups, with ** representing the mean. P The significance level is <0.0001.

[0034] Figure 3 Yeast extract activity test, including: A: Number of Trichogramma eggs per artificial egg; B: Larval survival rate; C: Prepupal rate; D: Pupation rate; E: Emergence rate; Error bars in the figure represent standard errors (SEM). One-way ANOVA and Tukey's test were used for comparisons between groups. Completely different letters on the bars indicate significant differences. p <0.05, meaning that the difference was not significant if the letters were the same. p >0.05). Detailed Implementation

[0035] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0036] The Trichogramma wasp described in this invention is the pine caterpillar Trichogramma wasp, obtained by propagating from rice moth eggs. The test population was raised in an artificial climate chamber under the following conditions: temperature 25±1℃, relative humidity 70%±5%, and photoperiod L14:D10. Female wasps used for introduction were selected from individuals that had emerged within 24 hours, had fully mated, and had not yet laid eggs.

[0037] In artificial cultivation, the culture medium is encapsulated in an artificial carrier to form a single-cell culture unit. When introducing bees, the bee-egg ratio is 8:1. The cells are cultured under the following conditions: temperature 25±1℃, relative humidity 75%±5%, photoperiod 14L:10D, and light intensity 30 Lux. The bees are removed 24 hours after introduction, and the cells are then cultured under shaded conditions. The egg stage, larval stage, prepupal stage, pupal stage, and emergence of Trichogramma are continuously recorded.

[0038] Example 1: Preparation and application of basic insect-free artificial culture medium 1. Culture medium composition The basic insect-free artificial culture medium described in this embodiment, i.e., the basic culture medium, is composed of the following components: 1 mL egg yolk, 1 mL TNM-FH insect cell culture medium, 0.3 mL Nissl salt solution, 0.1 mL trehalose solution (0.1 g / mL), and 0.1 mL penicillin-streptomycin mixture (100X) (containing 10000 u / mL penicillin and 10000 μg / mL streptomycin) TNM-FH medium was purchased from Wuhan Pronosai Life Science Technology Co., Ltd., TNM-FH insect medium (Pronosai).

[0039] 2. Group allocation method (1) Egg yolk treatment Take one fresh egg, disinfect the eggshell surface with 75% ethanol, and then place it in a clean bench for UV irradiation for 30 minutes. Under aseptic conditions, crack the egg, remove the egg white, keep the yolk, and use a sterile pipette to collect it for later use.

[0040] (2) Preparation of Nissl salt solution Weigh out 7.5 g of NaCl, 0.2 g of NaHCO3, 0.2 g of CaCl2, and 0.1 g of KCl. Add distilled water to a final volume of 100 mL, mix well, autoclave at 121℃ for 30 min, and store at 4℃ after natural cooling for later use.

[0041] (3) Preparation of trehalose solution Weigh 1 g of trehalose, add distilled water to a final volume of 10 mL, mix well, filter through a 0.2 μm filter membrane for sterilization, and store at -20℃ for later use.

[0042] 3. Culture medium preparation method Under aseptic conditions, 1 ml of egg yolk, 1 ml of TNM-FH insect cell culture medium, 0.3 ml of Nissl salt solution, 0.1 ml of trehalose solution, and 0.1 ml of penicillin-streptomycin mixture were added sequentially to a sterile centrifuge tube and mixed thoroughly to prepare 2.5 ml of basal culture medium, which is an artificial culture medium without silkworm hemolymph.

[0043] 4. Application Method The culture medium was encapsulated in an artificial carrier to make egg cards, which were then placed in a clean glass tube. Female Trichogramma wasps that had emerged within 24 hours, mated fully, and had not yet laid eggs were introduced at a wasp-to-egg ratio of 8:1. After 24 hours of inoculation in darkness, the wasps were removed, and the egg cards were transferred to a darkened environment for further cultivation. Before emergence, the carrier was appropriately opened to facilitate adult emergence.

[0044] 5. Implementation Results The basal culture medium described in this embodiment does not contain any insect-derived media and can serve as a foundational system for subsequent functional component optimization. This system can provide basic nutrition and ionic environment for the in vitro development of Trichogramma wasps, but there is still room for further optimization of subsequent developmental indicators.

[0045] Example 2: Insect-free artificial culture medium with added amino acid mixture and its application 1. Culture medium composition Based on the basic culture medium of Example 1, an amino acid mixture was added, with water as the solvent, and the formula is shown in Table 1.

[0046] Table 1. Amino acid mixture formulation β-alanine 238 L-Lysine hydrochloride 535 glycine 568 L-methionine 230 L-alanine 671 L-phenylalanine 117 L-arginine hydrochloride 476 L-proline 1385 L-Asparagine 0.9 L-serine 960 L-Cystine Dihydrochloride 2.4 L-threonine 868 L-glutamic acid 3761 L-Tryptophan 74 L-glutamine 2155 L-tyrosine disodium salt 384 L-histidine hydrochloride monohydrate 1071 L-valine 284 L-Isoleucine 425 ornithine 88 L-Leucine 330 2. Preparation method Under aseptic conditions, the basal culture medium was first prepared according to Example 1; then, 0.1 ml of amino acid mixture was added to the basal culture medium.

[0047] After thorough mixing, an insect-free artificial culture medium with added amino acids was obtained.

[0048] 3. Application Method The obtained culture medium was encapsulated, inoculated with wasps, and cultured according to the method in Example 1. During the culture process, the number of Trichogramma eggs, larval survival, and subsequent development status in each artificial culture unit were continuously recorded.

[0049] 4. Implementation Results The results showed that supplementing the basal culture medium with an amino acid mixture supported the survival of Trichogramma wasp eggs and larvae, indicating that these two amino acids are important active factors in the early development of Trichogramma wasps. This example laid a nutritional foundation for subsequent prepupal formation and further development.

[0050] Example 3: Insect-free artificial culture medium supplemented with B vitamins and its application 1. Culture medium composition Based on the culture medium of Example 2, a vitamin B mixture prepared according to the results of targeted metabolism was further added, with water as the solvent. The formulation of the vitamin B mixture is shown in Table 2: Table 2 Concentration of B vitamins in mixed solution Thiamine VB1 Vitamin B1 / Thiamine 703.8239214 Riboflavin VB2 Vitamin B2 / Riboflavin 12528.56045 Nicotinic acid VB3 Vitamin B3 / Niacin 1455.250656 Nicotinamide VPP Vitamin B3 / Vitamin PP / Niacinamide 165.5889118 Pantothenic acid VB5 Vitamin B5 / Pantothenic Acid 41546.43681 Pyridoxal VB6 Vitamin B6 / pyridoxal 0.274839447 Pyridoxal 5'-phosphate PLP Vitamin B6 / 5-pyridoxal phosphate 478.2480984 Biotin VB7 Vitamin B7 / Vitamin H / Coenzyme R / Biotin 647.6303127 Folic acid VB9 Vitamin B9 / Vitamin M / Folic Acid 73.69184619 Cyanocobalamin VB12 Vitamin B12 / Cyanocobalamin / Cobalamin 0.275097117 2. Preparation method First, a culture medium containing an amino acid mixture was prepared according to Example 2, and then 0.1 ml of a vitamin B mixture was added to it.

[0051] After thorough mixing, an insect-free artificial culture medium containing amino acids and B vitamins was obtained.

[0052] 3. Application Method The obtained culture medium was packaged, inoculated with bees, and cultured as described in Example 1. The development of Trichogramma wasps from larvae to prepupae was observed, and the prepupae rate and pupation status were recorded.

[0053] 4. Implementation Results The results showed that the prepupal rate of Trichogramma wasps increased from 55.96% to 83.46% after the addition of vitamin B mixture; at the same time, some individuals were able to continue developing and successfully pupate, with a pupation rate of 9.21%. This indicates that B vitamins have a significant promoting effect on the development of Trichogramma wasps from the egg-larval stage to the prepupal stage, and are an important functional component in the further optimization of insect-free culture media.

[0054] Example 4: Insect-free artificial culture medium with added yeast extract and its application 1. Culture medium composition Based on the culture medium of Example 3, yeast extract (Oxoid) was further added. TM Yeast extract powder).

[0055] The yeast extract solution has a concentration of 0.2 g / mL, and can be added in 20 μL, 40 μL or 60 μL as needed.

[0056] 2. Preparation method First, a culture medium containing amino acids and B vitamins was prepared according to Example 3. Then, a yeast extract solution was added and mixed thoroughly to obtain the final insect-free artificial culture medium.

[0057] 3. Application Method The obtained culture medium was packaged and inoculated and cultured with bees according to the conditions described in Example 1. The formation of prepupae, pupation and adult emergence of Trichogramma wasps were continuously observed, and the treatment groups with different amounts of yeast extract were compared.

[0058] 4. Implementation Results The results showed that supplementing the culture medium in Example 3 with yeast extract significantly improved the pupation and emergence of Trichogramma wasps. Compared with the treatment without yeast extract, the addition of yeast extract significantly increased both the pupation and emergence rates; under the conditions studied in this invention, the pupation rate reached a maximum of 82.44%, and the emergence rate reached a maximum of 27.22%. This indicates that yeast extract can further improve the nutritional and metabolic support conditions required for the later development of Trichogramma wasps, playing an important role in establishing a complete insect-free artificial culture system.

[0059] Example 5: Comparative Analysis of Trichogramma wasps on Two Artificial Culture Media 4.1 Amino Acid Test CK+AA: CK culture medium with added mixed amino acids prepared in Example 2; CK: The basal culture medium prepared in Example 1; like Figure 1 As shown, in vitro supplementation with an amino acid mixture significantly increased the egg production and survival rate of Trichogramma wasps (egg production: t 24 =7.641, P <0.0001; Survival rate: t 24 =7.328, P <0.0001. The egg production figure indicates that the added amino acid mixture significantly promotes the development of Trichogramma wasp larvae.

[0060] 4.2 B Vitamin Test Control group CK + AA was based on a mixture of basal culture medium and amino acids; The treatment group CK+AA+VB was prepared by culturing Trichogramma wasps in a culture medium containing a CK+AA+vitamin B mixture (prepared based on the results of targeted metabolism) as described in Example 3.

[0061] Figure 3 The results showed that the addition of the VB mixture (vitamin B complex 1-12) significantly promoted the rate of Trichogramma wasps developing into prepupae.

[0062] Regarding egg production, the control group (CK+AA) averaged 116 eggs, while the treatment group (CK+AA+VB) averaged 111.7 eggs, with no significant difference between the two groups (t). 18 = 0.79, P = 0.44)( Figure 2 (A) indicates that the optimized culture medium had no significant effect on oviposition stability. However, the prepupae rate in the treatment group reached 83.46%, significantly higher than the 55.96% in the control group (t). 18 = 6.92, P <0.001)( Figure 2 (B) indicates that the addition of the vitamin mixture mainly promoted the developmental stage of Trichogramma wasp larvae, increasing the rate of prepupae. Furthermore, some Trichogramma wasps successfully pupated after the addition of the VB mixture. The pupation rate was 9.21%, significantly higher than that of the CK+AA group (t...). 18 = 12.049, P <0.001). Therefore, adding the VB mixture can promote the pupation of Trichogramma wasps ( Figure 2 C).

[0063] 4.3 Yeast extract test The above results indicate that the mechanism promoting pupation and emergence in *Trichogramma rubrum* is not a single compound, and most likely not a unique active factor of *Bombyx mori* hemangioma, but rather a functional module. Screening and tracking such a module is extremely complex. In this experiment, yeast extracts at different gradients were added to the CK+AA+VB base to replace the functional module promoting pupation.

[0064] CK+AA+VB: basal culture medium + amino acid mixture + B vitamin mixture; Yeast 20μl: CK+AA+VB+Yeast extract (0.2g / mL) 20 μl; Yeast 40μl: CK+AA+VB+Yeast extract (0.2g / mL) 40 μl; Yeast 60μl: CK+AA+VB+Yeast extract (0.2g / mL) 60 μl.

[0065] like Figure 3 The results showed that the treatment groups with added yeast of 20 μl, 40 μl and 60 μl had significantly lower oviposition rates (One-way ANOVA, F(3, 51) = 0.126, p = 0.945). Figure 3 A) Survival rate (One-way ANOVA, F) (3, 51) = 1.026, P = 0.39; Figure 3 B) and prepupae rate (One-way ANOVA, F)(3, 51) = 1.531, P = 0.22; Figure 3 C), and CK+AA+VB, showed no significant difference. However, the pupation rate (One-way ANOVA, F) was significantly different. (3, 51) = 169.386, P <0.0001; Figure 3 D) and feathering rate (One-way ANOVA, F) (3, 51) = 71.312, P <0.0001; Figure 3 In E), the 20 μl, 40 μl, and 60 μl treatment groups were all higher than the control group CK+AA+VB. This indicates that the yeast extract can indeed metabolize the module in the hemolymph of silkworms that promotes the pupation of Trichogramma wasps. This further verifies that the culture medium formulation of this invention can replace natural insect-derived materials, providing a suitable developmental environment and sufficient developmental substrate.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An artificial culture medium for Trichogramma wasps without insect media, characterized in that, The artificial culture medium for Trichogramma bees includes a basal culture medium, which comprises: egg yolk, TNM-FH medium, Nissl salt solution, trehalose solution, and a penicillin-streptomycin mixture; the artificial culture medium for Trichogramma bees also includes an amino acid mixture, B vitamins, and yeast extract.

2. The artificial culture medium for Trichogramma wasps according to claim 1, characterized in that, The volume ratio of the egg yolk, TNM-FH insect cell culture medium, Nissl salt solution, 0.1 g / mL trehalose solution, and penicillin-streptomycin mixture in the basal culture medium is 1:1:0.3:0.1:0.

1.

3. The artificial culture medium for Trichogramma according to claim 1, characterized in that, Nissl salt solution is prepared by adding 7.5g NaCl, 0.2g NaHCO3, 0.2g CaCl2, and 0.1g KCl to distilled water and bringing the volume to 100 mL.

4. The artificial culture medium for Trichogramma according to claim 1, characterized in that, The penicillin-streptomycin mixture contains 10,000 u / ml of penicillin and 10,000 μg / ml of streptomycin, and the penicillin-streptomycin mixture is added in equal volume to the trehalose solution.

5. The artificial culture medium for Trichogramma according to claim 1, characterized in that, The amino acid mixture was prepared into an amino acid mixed solution and then added to the artificial culture medium of Trichogramma wasps. The concentrations of each amino acid in the amino acid mixed solution were as follows: 。 6. The artificial culture medium for Trichogramma wasps according to claim 1, characterized in that, In the artificial culture medium for Trichogramma bees, the volume ratio of the amino acid mixture solution to the basal culture medium is 1:

25.

7. The artificial culture medium for Trichogramma according to claim 1, characterized in that, In the artificial culture medium for Trichogramma bees, B vitamins were prepared into a mixed B vitamin solution and then added to the culture medium. The concentrations of each B vitamin in the mixed B vitamin solution were as follows: thiamine 703±10 ng / mL, riboflavin 12528±10 ng / mL, niacin 1455±10 ng / mL, nicotinamide 165±10 ng / mL, pantothenic acid 41546±10 ng / mL, pyridoxal 0.27±0.03 ng / mL, pyridoxal 5-phosphate 478±10 ng / mL, biotin 647±10 ng / mL, folic acid 73±10 ng / mL, and cobalamin 0.27±0.03 ng / mL.

8. The artificial culture medium for Trichogramma according to claim 7, characterized in that, In the artificial culture medium for Trichogramma bees, the volume ratio of the B vitamin mixture solution to the basal culture medium is 1:

25.

9. The artificial culture medium for Trichogramma according to claim 1, characterized in that, The yeast extract was prepared into a yeast extract solution and then added. The concentration of the yeast extract solution was 0.2 g / mL. In the artificial culture medium for Trichogramma bees, the volume ratio of the amount of yeast extract solution added to the volume of the basal culture medium was (0.2-0.6):

25.

10. The application of the artificial culture medium for Trichogramma as described in claim 1 in the in vitro culture process during the artificial breeding of Trichogramma.

Images