A sterile artificial feed for onion flies, its preparation method and application

Abstract

This invention relates to the field of insect feed technology, specifically to a sterile artificial feed for onion flies, its preparation method, and its application. The sterile artificial feed for onion flies is prepared by mixing sterilized raw materials, including: semi-fermented onion filtrate, semi-fermented onion residue, choline chloride, L-ascorbic acid, agar powder, TSB medium, and water. The semi-fermented onion filtrate includes fermented onion filtrate and unfermented onion filtrate, and the semi-fermented onion residue includes fermented onion residue and unfermented onion residue. The fermented onion residue and fermented onion filtrate are respectively the solid-phase product and liquid-phase product of the mixture of onion grinding liquid and onion fly larvae grinding liquid after fermentation. The sterile artificial feed for onion flies provided by this invention, after semi-fermentation treatment, can meet the growth requirements of sterile onion flies, allowing them to maintain a sterile state for full-stage development.

Description

Technical Field

[0001] This invention relates to the field of insect feed technology, specifically to a sterile artificial feed for onion flies, its preparation method, and its application. Background Technology

[0002] The garlic fly, also known as the onion maggot, is a major pest of onions and other lily family crops worldwide. Its larvae damage the bulbs of onions and other lily family crops, causing huge economic losses. If not effectively controlled, it can lead to crop losses of 50% to 100% for onions, garlic, scallions, and leeks.

[0003] Insects are closely intertwined with their gut microbiota. Most insects contain a variety of symbiotic bacteria in their gut, which rely on nutrients provided by the host for survival. Studies have shown that the primary function of gut symbiotic flora is to provide essential nutrients, followed by functions related to digestion and detoxification. In some cases, gut bacteria can serve as a microbial resource for insect control, making it crucial to study the performance and specific functions of individual gut bacteria within insects. Therefore, preparing sterile test insects is particularly important for investigating the interactions between specific bacterial strains and insects.

[0004] Currently, the conventional method involves sterilizing insect eggs and adding antibiotics to the food of larvae and adults to cultivate sterile insects. However, this method often leads to changes in the physiological and biochemical state of the insects due to the toxicity of the antibiotics themselves, resulting in decreased adaptability. Other methods, such as ultraviolet disinfection, can also produce sterile artificial feed. However, for oligophagous pests like onion flies, conventional sterile artificial feed is insufficient to guarantee the rearing of sterile onion flies with normal physiological and biochemical states. Summary of the Invention

[0005] To address the technical problem that existing antibiotic-containing feeds cannot meet the needs of aseptic onion fly larvae for complete development from instar to adult, this invention provides a sterile artificial feed, its preparation method, and its application. Feeding aseptic onion fly larvae with this artificial feed results in aseptic onion flies with normal physiological and biochemical states, providing high-quality experimental material for studying the interaction between bacterial strains and onion flies.

[0006] In a first aspect, the present invention provides a sterile artificial feed for onion flies. The sterile artificial feed for onion flies is prepared by mixing sterilized raw materials, including: semi-fermented onion filtrate, semi-fermented onion residue, choline chloride, L-ascorbic acid, agar powder, TSB medium, and water; wherein the semi-fermented onion filtrate includes fermented onion filtrate and unfermented onion filtrate, and the semi-fermented onion residue includes fermented onion residue and unfermented onion residue. The fermented onion residue and fermented onion filtrate are respectively the solid-phase product and liquid-phase product of the mixture of onion grinding liquid and onion fly larvae grinding liquid after fermentation.

[0007] Furthermore, the raw materials include: 5-10g fermented onion residue, 5-10g unfermented onion residue, 80-100ml fermented onion filtrate, 80-100ml unfermented onion filtrate, 0.2-0.5g choline chloride, 0.5-1.0g L-ascorbic acid, 2-3g agar powder, 5-8g TSB medium, and 100-150ml water.

[0008] Furthermore, the raw materials include: 5g fermented onion residue, 5g unfermented onion residue, 80ml fermented onion filtrate, 80ml unfermented onion filtrate, 0.24g choline chloride, 0.56g L-ascorbic acid, 2g agar powder, 6g TSB medium, and 103ml water.

[0009] Secondly, the present invention provides a method for preparing sterile artificial feed for onion flies, comprising the following steps:

[0010] (1) Add sterile water to the white part of the scallion and grind it into a paste to obtain scallion grinding liquid. Then grind the larvae of the onion fly to obtain onion fly larvae grinding liquid. Mix the scallion grinding liquid and the onion fly larvae grinding liquid and place them in a shaker incubator. Ferment for 12-24 hours at 25-37℃ and 180-230rpm. After filtering with gauze, obtain fermented scallion residue and initial fermented scallion filtrate. The initial fermented scallion filtrate is then sterilely filtered to obtain fermented scallion filtrate.

[0011] (2) Add sterile water to the white part of the scallion and grind it into a paste to obtain scallion grinding liquid. After filtering with gauze, unfermented scallion residue and initial unfermented scallion filtrate are obtained. The initial unfermented scallion filtrate is sterile filtered to obtain unfermented scallion filtrate.

[0012] (3) Choline chloride and L-ascorbic acid were added to water and mixed to dissolve, and then filtered aseptically to obtain mixture A;

[0013] (4) Add agar powder and TSB medium to water and stir evenly to obtain medium B. High pressure steam sterilize medium B, fermented onion residue and unfermented onion residue.

[0014] (5) In a sterile environment, fermented onion residue, unfermented onion residue, fermented onion filtrate, unfermented onion filtrate, mixed liquid A and culture medium B are mixed evenly to obtain sterile artificial feed for onion flies.

[0015] Furthermore, in step (1), the onion grinding liquid and the onion fly larvae grinding liquid are mixed and placed in a shaker incubator for fermentation at 25°C and 180 rpm for 12 hours.

[0016] Furthermore, in step (1), the number of onion fly larvae is 10-20.

[0017] Furthermore, the filter membrane pore size in steps (1), (2), and (3) for sterile filtration is 0.22 μm.

[0018] Thirdly, the present invention provides an application of sterile artificial feed for onion flies in the rearing of sterile onion flies.

[0019] The beneficial effects of this invention are as follows:

[0020] Onion flies require the aid of intestinal microorganisms for digestion and absorption of their food to meet their growth and development needs. However, sterile onion flies lack the necessary microbial environment for survival during their growth and development. Experiments by the inventors revealed that sterile onion fly larvae fed unfermented food experience slow growth or even poisoning and death, while feeding them fully fermented food leads to malnutrition, resulting in slow growth or even death. The sterile onion fly artificial feed provided by this invention, through semi-fermentation, meets the nutritional needs of sterile onion flies, allowing them to maintain a sterile state for full-stage development. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 These are colony images of intestinal homogenate of onion fly larvae on TSA and PDA agar plates. (A) Colony image of intestinal homogenate of sterile onion fly larvae spread on TSA. (B) Colony image of intestinal homogenate of normal onion fly larvae spread on TSA. (C) Colony image of intestinal homogenate of sterile onion fly larvae spread on PDA. (D) Colony image of intestinal homogenate of normal onion fly larvae spread on PDA.

[0023] Figure 2 These are molecular detection patterns of sterile onion fly larvae. (A) is a 16S rRNA electrophoresis pattern, and (B) is an ITS electrophoresis pattern. In the pattern, M = molecular weight marker, NC = negative control, NF = normal feeding, and AF = sterile feeding.

[0024] Figure 3 This is a bar chart showing the survival rate of second-instar larvae of onion flies fed with different feeds.

[0025] Figure 4 This is a bar chart showing the pupation rate of onion fly larvae fed with different feeds.

[0026] Figure 5 This is a bar chart showing the percentage of the total weight of pupae and larvae of onion flies when fed different feeds.

[0027] Figure 6 This is a bar chart showing the pupal emergence rate of onion flies fed with different feeds. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0029] The bacteria mentioned in this invention as sterile onion flies refer to extracellular symbiotic bacteria in the intestines or on the surface of the onion fly, excluding intracellular symbiotic bacteria present in the onion fly eggs.

[0030] Example 1

[0031] Aseptic onion fly artificial feed formula: 5g fermented onion residue, 5g unfermented onion residue, 80ml fermented onion filtrate, 80ml unfermented onion filtrate, 0.24g choline chloride, 0.56g L-ascorbic acid, 2g agar powder, 6g TSB medium, and 103ml deionized water.

[0032] Preparation method:

[0033] (1) Peel off the outer layers of the scallion and discard the leaves. Keep the white part of the scallion, wash it with sterile water, and rinse it three times. Use scissors sterilized with 75% ethanol solution to cut the white part of the scallion into small pieces of 1-2 cm. Weigh 50 g of chopped scallion and put it into a food processor. Add 50 ml of sterile water and grind for 2 minutes each time, for a total of five times. Grind all the scallion pieces into a paste to obtain scallion grinding liquid. Use tweezers to transfer 10 onion fly larvae into a centrifuge tube containing 10 ml of 1×PBS and grind them with a disposable pestle for about 5 minutes to obtain onion fly larvae grinding liquid. Pour the scallion grinding liquid and onion fly larvae grinding liquid into a clean 500 ml Erlenmeyer flask. Cover the mouth of the flask with two layers of transparent sealing film, and then place the Erlenmeyer flask in a shaker incubator for 12 hours of fermentation at 25°C and 180 rpm. Cut seven layers of square gauze with a side length of 10 cm and stack them together to form a simple filter device. Slowly pour the fermented onion liquid onto a piece of cheesecloth, squeezing out the filtrate into a new 500 ml Erlenmeyer flask as the initial fermented onion filtrate. Wrap the remaining fermented onion residue on the cheesecloth with aluminum foil for later use. Filter the initial fermented onion filtrate obtained in the previous step using a vacuum pump and a Buchner funnel. Place filter paper moistened with deionized water on the Buchner funnel, then pour in the filtrate and perform three vacuum filtrations, collecting the filtrate in a 500 ml Erlenmeyer flask. Filter again using a 0.22 μM filter flask and a vacuum pump. Tighten the cap of the vacuum flask on a clean bench to obtain the fermented onion filtrate.

[0034] (2) Repeat step (1) to obtain unfermented onion residue and unfermented onion filtrate. The difference is that it is not necessary to add onion fly larvae for fermentation.

[0035] (3) Weigh the prescribed amounts of choline chloride and L-ascorbic acid into a 10 mL centrifuge tube. Add 3 mL of deionized water and shake until completely dissolved. On a clean bench, use a new 5 mL syringe connected to three 0.22 μM syringe filters to filter and sterilize the solution to obtain mixture A. Then, place it into a sterile 10 mL centrifuge tube for later use.

[0036] (4) Weigh the prescribed amount of agar powder and TSB medium into a 500 mL Erlenmeyer flask, and then add 100 mL of deionized water to prepare the medium. Rinse the glass rod with deionized water and use it to stir the mixture until homogeneous to obtain medium B. Then seal the Erlenmeyer flask with two layers of transparent sealing film. Wrap the previously mentioned fermented onion residue and unfermented onion residue with aluminum foil, and sterilize them together with medium B in an autoclave at 121°C for 20 minutes.

[0037] (5) On a clean bench, pour the sterilized mixture A, fermented onion filtrate, and unfermented onion filtrate into culture medium B, and gently rotate to mix thoroughly. Finally, add the fermented onion residue and unfermented onion residue, and shake thoroughly by hand to obtain sterile artificial feed for onion flies.

[0038] Example 2

[0039] Aseptic onion fly artificial feed formula: 10g fermented onion residue, 10g unfermented onion residue, 100ml fermented onion filtrate, 10ml unfermented onion filtrate, 0.5g choline chloride, 1.0g L-ascorbic acid, 2g agar powder, 5g TSB medium, 103ml deionized water.

[0040] Preparation method:

[0041] The preparation method is the same as in Example 1, except that the amount of aseptic artificial feed formula is different. In step (1), the amount of onion fly larvae added is 20, and the fermentation is carried out at 37°C and 230 rpm for 24 hours.

[0042] Example 3

[0043] Aseptic onion fly artificial feed formula: 5g fermented onion residue, 5g unfermented onion residue, 100ml fermented onion filtrate, 100ml unfermented onion filtrate, 0.5g choline chloride, 1.0g L-ascorbic acid, 2g agar powder, 5g TSB medium, and 103ml deionized water.

[0044] Preparation method:

[0045] The preparation method is the same as in Example 1, except that the amount of aseptic artificial feed formulation is different.

[0046] Comparative Example 1

[0047] Aseptic onion fly artificial feed formula: 10g fermented onion residue, 160ml fermented onion filtrate, 0.24g choline chloride, 0.56g L-ascorbic acid, 2g agar powder, 6g TSB medium, and 103ml deionized water.

[0048] Preparation method:

[0049] The preparation method is the same as in Example 1, except that step (2) is not required.

[0050] Comparative Example 2

[0051] Aseptic onion fly artificial feed formula: 10g unfermented onion residue, 160ml unfermented onion filtrate, 0.24g choline chloride, 0.56g L-ascorbic acid, 2g agar powder, 6g TSB medium, and 103ml deionized water.

[0052] Preparation method:

[0053] The preparation method is the same as in Example 1, except that step (1) is not required.

[0054] Comparative Example 3

[0055] Aseptic onion fly artificial feed formula: 5g fermented onion residue, 5g unfermented onion residue, 80ml fermented onion filtrate, 80ml unfermented onion filtrate, 0.24g choline chloride, 0.56g L-ascorbic acid, 2g agar powder, 6g TSB medium, 103ml deionized water, and antibiotics with final concentrations of 10mg / mL nystatin, 10mg / mL actinomycin, 10mg / mL penicillin, and 10mg / mL streptomycin sulfate.

[0056] Preparation method:

[0057] The preparation method is the same as in Example 1, except that the aseptic treatment method is changed to: adding fermented onion residue and unfermented onion residue in step (5) and then adding antibiotics, and shaking thoroughly by hand to prepare semi-fermented feed with added antibiotics.

[0058] Experiment Example 1: Complete Preparation of Aseptic Onion Fly Larvae

[0059] (1) Preparation of sterile onion fly eggs

[0060] Collect onion fly eggs using moist sand containing garlic cloves. Pour the sand and eggs into water and filter through a 100-mesh sieve; the eggs will remain on the sieve. Allow the eggs to dry naturally on the sieve, then gently brush them onto a culture dish. Transfer the collected eggs to a sterile cell strainer and place them on a laminar flow hood for later use.

[0061] Place the cell sieve containing the eggs into a culture dish containing NaClO. Immerse in a 0.26% NaClO solution for 0.5 minutes. Then transfer the cell sieve to a culture dish containing EtOH and immerse it in a 75% EtOH solution for another 0.5 minutes. Repeat this process three times, alternating between 0.26% NaClO and 75% EtOH every 0.5 minutes. After this, sterile onion fly eggs will be obtained.

[0062] (2) Rearing of sterile onion fly larvae

[0063] Using scissors sterilized with an alcohol lamp, cut off the end of a 1 ml pipette tip. Ensure the cut end of the tip is larger than the diameter of the eggs (approximately 2 mm). Then, using a pipette, transfer the eggs (currently in EtOH solution) from the EtOH solution to a centrifuge tube containing the artificial sterile feed from Example 1. Each centrifuge tube should contain approximately 20-50 eggs. Use a pipette to remove excess ethanol from the centrifuge tube. Place the capped centrifuge tube in a resealable bag, placing a cotton ball at the bag opening for ventilation. Finally, place the bag in an incubator at 25°C (relative humidity: 50%-70%; photoperiod: 16 hours light: 8 hours dark) for observation and incubation. After approximately three days, the sterile onion fly eggs in the feed will begin to hatch. The hatched larvae will become active, and the surface of the sterile feed will no longer be smooth, allowing the larvae to begin feeding on it. Continue to observe the larvae until they enter the second instar larval stage. During this period, continuously monitor their growth and development. Nine to twelve days later, more than 80% of the eggs had developed into third-instar larvae. This indicates that onion fly larvae can successfully grow and develop.

[0064] Experimental Example 2: Plate culture detection of sterile onion fly larvae

[0065] Using the sterile onion fly larvae raised in Experiment 1, three sterile third-instar larvae were randomly selected from a centrifuge tube. They were then transferred to a petri dish containing sterile water, and the artificial feed was rinsed off. Using sterilized dissecting forceps, the larvae were grasped, and the head and tail were removed using dissecting scissors. The tail of the larva was grasped with forceps, and the internal organs were gently squeezed from the tail towards the head with another forceps. The internal organs were placed in deionized water, and the intestine was gently removed by pressing the adipose tissue with forceps. The extracted intestine was then placed in sterile water for later use. The extracted intestinal tissue was placed in a sterile 1.5 mL centrifuge tube, and 500 μL of sterile 1x PBS buffer was added. The intestinal tissue was then homogenized using a disposable grinder. 100 μL of the mixture was added to nutrient agar medium and spread on TSA and PDA plates using an L-shaped spreader. The culture dishes were placed in a biochemical incubator and incubated at 28 degrees Celsius for 72 hours to observe the growth of the colonies. In addition, conventionally cultured onion flies (without sterile treatment of their eggs and fed with unsterilized artificial feed) were used as a control group for plating.

[0066] The result is Figure 1 It is known that no fungal or bacterial colonies were isolated from the intestinal tissue slurry of onion fly larvae that were fed with the aseptic artificial feed provided by the present invention and subjected to aseptic culture. This indicates that the aseptic artificial feed provided by the present invention has a good aseptic treatment effect and can be used as feed for raising aseptic onion flies.

[0067] Experiment Example 3: Molecular detection of sterile onion fly larvae

[0068] Sterile onion fly larvae reared in Experiment 1 were used. Total DNA was extracted from the intestinal tissue of the sterile onion fly larvae, and the DNA concentration was measured using a UV spectrophotometer. Normal feeding involved feeding unsterilized eggs with infected onions, resulting in infected onion fly larvae under sterile conditions. Total DNA was extracted from their intestinal tissue for testing. The negative control was achieved using ultrapure water during the testing process.

[0069] PCR was performed using universal primers for bacterial 16S rRNA (1492R, 27F). The total reaction volume was 25 μL, including 1 μL DNA template, 0.5 μL forward primer, 0.5 μL reverse primer, 10.5 μL ddH2O, and 12.5 μL 2x Taq PCRMaster Mix. PCR conditions were as follows: initial denaturation at 94°C for 3 minutes; 35 cycles, each consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 90 seconds; final extension at 72°C for 10 minutes. PCR products were stored at 4°C for further analysis.

[0070] PCR was performed using universal fungal ITS primers (ITS1, ITS4). The total reaction volume was 25 μL, including 1 μL DNA template, 0.5 μL forward primer, 0.5 μL reverse primer, 10.5 μL ddH2O, and 12.5 μL 2x Taq PCR MasterMix. PCR conditions were set as follows: 95°C for 5 minutes; 35 cycles, each consisting of 95°C for 30 seconds, 55°C for 1 minute, and 72°C for 90 seconds; and a final reaction at 72°C for 10 minutes. PCR products were stored at 4°C for further analysis.

[0071] Super Green nucleic acid dye was uniformly mixed with both types of PCR products and analyzed by electrophoresis on a 1% agarose gel in 1x TAE buffer. 3 μL of DNA molecular weight marker was used as a reference. The gel was observed using a UV transilluminator to locate the target fragment.

[0072] The results showed that PCR analysis based on bacterial 16S rRNA revealed a band at approximately 1500 bp. Figure 2 (A) was identified as an intracellular symbiotic bacterium. Wolbachia (The GenBank accession number for this nucleotide sequence is OR564190). However, no bands were observed in PCR analysis targeting the fungal ITS region. Figure 2 (B)). This shows that the onion fly has reached a sterile state.

[0073] Experiment Example 4: Effects of different sterile artificial feeds for onion flies on their growth and development

[0074] Sterile eggs were prepared according to the method in Experimental Example 1 and fed with the sterile artificial feed for onion flies of Examples 1, 1, 2, and 3, respectively. Additionally, a group of unsterilized onion fly eggs was fed with unsterilized chopped scallions as a negative control.

[0075] Five tubes were prepared for each type of feed as five replicates, with 20 sterile larvae eggs in each tube. The developmental status of the larvae in the tubes was observed daily. Using the eggs fed with scallions as the developmental indicator, the growth and development of all larvae were observed. The survival rate, pupation rate, emergence rate, and pupal weight of the second-instar larvae fed with other feeds were recorded when the normally fed larvae reached the second instar, pupated, and emerged as adults.

[0076] In this experiment, the aseptic semi-fermented feed was the aseptic artificial feed for onion flies in Example 1, the aseptic fully fermented feed was the aseptic artificial feed for onion flies in Comparative Example 1, the aseptic non-fermented feed was the aseptic artificial feed for onion flies in Comparative Example 2, the semi-fermented feed with antibiotics was the aseptic artificial feed for onion flies in Comparative Example 3, and the normal feeding with bacteria was the negative control.

[0077] result:

[0078] Depend on Figure 3 It is known that almost all eggs can develop to the second instar when fed normally, and almost all sterile onion fly larvae fed with sterile semi-fermented feed also develop to the second instar. When sterile onion fly eggs are fed with sterile fully fermented feed and semi-fermented feed plus antibiotics, more than 70% of the larvae can develop to the second instar. However, when onion flies are fed with sterile unfermented feed, nearly 80% of the larvae cannot develop to the second instar and die.

[0079] Similar to the results above, Figure 4 This indicates that, except for about 20% of sterile eggs fed with sterile, unfermented feed, over 80% of larvae fed with other feeds were able to pupate. However, from Figure 5 It can be seen that the pupae of sterile insect eggs fed with sterile semi-fermented feed after pupation weighed approximately the same as those of infected onion fly pupae, while the pupae raised using other methods weighed only 8%-30% of the infected pupae, indicating that these pupae did not develop normally. Furthermore, from Figure 6 It can be seen that among the pupating larvae mentioned above, all the onion fly pupae fed with sterile semi-fermented feed and those fed normally were able to emerge as adults, while the pupae obtained from the other feed groups were almost all unable to emerge.

[0080] The above results indicate that unfermented, fully fermented, and antibiotic-treated artificial feeds (aseptic artificial feeds for onion flies in Comparative Examples 1, 2, and 3) are all insufficient to guarantee the normal development of sterile onion flies. However, the aseptic semi-fermented feed provided by this invention can produce sterile onion fly adults with normal physiological and biochemical states, and the survival rate of onion flies is comparable to that of normal onion fly adults with bacteria.

[0081] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A sterile artificial feed for onion flies, characterized in that, The aseptic onion fly artificial feed is prepared by mixing sterilized raw materials, including: 5-10g fermented onion residue, 5-10g unfermented onion residue, 80-100ml fermented onion filtrate, 80-100ml unfermented onion filtrate, 0.2-0.5g choline chloride, 0.5-1.0g L-ascorbic acid, 2-3g agar powder, 5-8g TSB medium, and 100-150ml water. The preparation method of sterile artificial feed for onion flies includes the following steps: (1) Add sterile water to the white part of the scallion and grind it into a paste to obtain scallion grinding liquid. Then grind the larvae of the onion fly to obtain onion fly larvae grinding liquid. Mix the scallion grinding liquid and the onion fly larvae grinding liquid and place them in a shaker incubator. Ferment for 12-24 hours at 25-37℃ and 180-230rpm. After filtering with gauze, obtain fermented scallion residue and initial fermented scallion filtrate. The initial fermented scallion filtrate is then sterilely filtered to obtain fermented scallion filtrate. (2) Add sterile water to the white part of the scallion and grind it into a paste to obtain scallion grinding liquid. After filtering with gauze, unfermented scallion residue and initial unfermented scallion filtrate are obtained. The initial unfermented scallion filtrate is sterile filtered to obtain unfermented scallion filtrate. (3) Choline chloride and L-ascorbic acid were added to water and mixed to dissolve, and then filtered aseptically to obtain mixture A; (4) Add agar powder and TSB medium to water and stir evenly to obtain medium B. High pressure steam sterilize medium B, fermented onion residue and unfermented onion residue. (5) In a sterile environment, fermented onion residue, unfermented onion residue, fermented onion filtrate, unfermented onion filtrate, mixed liquid A and culture medium B are mixed evenly to obtain sterile artificial feed for onion flies.

2. The aseptic artificial feed for onion flies as described in claim 1, characterized in that, Raw materials include: 5g fermented onion residue, 5g unfermented onion residue, 80ml fermented onion filtrate, 80ml unfermented onion filtrate, 0.24g choline chloride, 0.56g L-ascorbic acid, 2g agar powder, 6g TSB medium, 103ml water.

3. The aseptic artificial feed for onion flies as described in claim 1, characterized in that, In step (1), the onion grinding liquid and the onion fly larvae grinding liquid are mixed and placed in a shaker incubator for fermentation at 25°C and 180 rpm for 12 hours.

4. The aseptic artificial feed for onion flies as described in claim 1, characterized in that, The number of onion fly larvae in step (1) is 10-20.

5. The aseptic artificial feed for onion flies as described in claim 1, characterized in that, The pore size of the filter membrane used for sterile filtration in steps (1), (2), and (3) is 0.22 μm.

6. The application of the aseptic artificial feed for onion flies as described in claim 1 in the rearing of aseptic onion flies.

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