CN113243339A

CN113243339A - Simple, convenient and efficient preparation method of aseptic fruit flies

Info

Publication number: CN113243339A
Application number: CN202110466175.1A
Authority: CN
Inventors: 汪肖云; 周帆
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2021-08-13
Anticipated expiration: 2041-04-28
Also published as: CN113243339B

Abstract

The invention belongs to the technical field of insect breeding, and particularly relates to a simple, convenient and efficient sterile fruit fly preparation method, which technically optimizes a fruit fly egg collecting device, a sterile culture medium preparation, egg rinsing sterilization, sterile culture and other aspects, for example, a sealing film commonly used in tissue culture technology is used for sealing a culture bottle/tube, can resist the high temperature of 135 ℃, can perform damp-heat sterilization, can ensure air exchange, and can achieve the purpose of sealing and preventing bacteria. Generally, the method can create a sterile and breathable growth environment for the growth of the fruit flies, can ensure the normal growth of the fruit flies, can greatly reduce the probability of contamination in the culture process, has the advantages of simple and convenient method, easy operation, low cost, good repeatability, high success rate, large-scale preparation and the like, and has important application value in the research of host-microorganism interaction in the field of life science.

Description

Simple, convenient and efficient preparation method of aseptic fruit flies

Technical Field

The invention belongs to the technical field of insect breeding, and particularly relates to a simple, convenient and efficient preparation method of aseptic fruit flies.

Background

Animals self-birth is closely associated with microorganisms that affect various aspects of the animal's physiological activities. Research shows that intestinal microflora can influence the correlation between host brain-intestinal axes, resist pathogen, stimulate inflammation, activate immune system and midgut regeneration, etc., so that how to regulate and apply microflora becomes a new technological field with great prospect. In recent years, Drosophila melanogaster (Drosophila melanogaster) has received increasing attention as a model organism for studying gut-host microbial interactions. The gut of drosophila and mammals shares many similarities in physiological structure, development, metabolism, and immunity, for example, the immune metabolic pathways that maintain gut homeostasis, gut function and integrity, and the gut signaling pathways that regulate host bacterial interactions are highly conserved. Research reports have shown that the drosophila melanogaster is used as a model organism for researching intestinal infection and pathology, recognition and defense mechanisms of intestinal pathogens, natural intestinal flora and disorder, differentiation and regeneration of intestinal cells, inflammatory bowel diseases and the like, and intestinal flora such as vibrio cholerae, pseudomonas aeruginosa, enterococcus faecalis and the like. In addition, a great deal of research shows that the drosophila is taken as a research model, and the drosophila has good application value for researching the relationship between intestinal microorganisms and prolonging the service life, the activity, regulation, metabolism and development stability of intestinal stem cells, dietary habits and the like.

Organisms in nature often carry a large number of microorganisms, and this natural microbial community is complex and diverse, and brings great importance to the study of host-microorganism interactions. Therefore, in order to investigate the details of host-microbial interactions, artificial control of the Drosophila's microbiota is required. Among them, artificial feeding of sterile fruit flies in the laboratory has become an ideal embodiment. The fruit fly can be brought to a sterile state by removing the outermost chorion of the fruit fly embryo and transferring the embryo to sterile food in a sterile environment. The aseptic fruit fly has no intestinal microbiota or colonization of other kinds of microorganisms, and can be used as a flexible and practical animal model for exploring the existence of microorganisms or the interaction relationship of specific microorganism species with a host under a single or multiple combined states and disclosing a complex interaction mechanism between bacteria and the host.

At present, although some sterile fruit fly models are prepared, the preparation methods still have some defects in practical operation: (1) therefore, the closed culture tube is used for preventing external bacteria from being polluted, so that the survival rate of the fruit flies is greatly reduced, and even the fruit flies cannot grow normally. (2) In order to inhibit the growth of flora, antibiotics are fed until the fruit flies grow out after the fruit flies hatch, but the method only can remove part of bacteria, and the problems of toxicity, drug effect, generated drug resistance and the like of the antibiotics are also considerable problems. (3) The fruit fly bottle plug with small aperture is used for achieving a certain 'bacteria blocking' effect, the plug can be used for preventing mites, but the bacteria blocking effect of the plug cannot be guaranteed, the plug can have the phenomena of yellow color, soft texture and large aperture after high-temperature sterilization, and whether bacteria blocking is uncertain or not can be determined. (4) The obtained aseptic fruit flies have low hatchability, high mortality rate, complicated preparation process and easy contamination, and the obtained aseptic fruit flies have limited number and are difficult to meet the requirements of subsequent researches. (5) The identification method of the sterile fruit fly model is single and has low credibility, for example, the method is used for researching whether the sterile fruit fly egg coating plate is used for judging whether the model is successfully constructed by observing whether a colony grows out or not, but the method does not consider the problem that whether the germ is infected or not in a half-moon growth period after the eggs are hatched. Therefore, there is a need to develop a simple and efficient method for preparing aseptic fruit flies, so as to increase the success rate of the method.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a simple, convenient and efficient preparation method of aseptic fruit flies, which has the advantages of simple and convenient method, easy operation, low cost, good repeatability, high success rate, large-scale preparation and the like, and has important application value in the research of the field of life science.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a simple, convenient and efficient preparation method of aseptic fruit flies, which comprises the following steps:

s1, preparing a sterile culture medium: weighing 5% of yeast, 10% of glucose, 1.2% of agar and 0.2% of potassium sorbate, adding water, mixing uniformly, heating and boiling, then sterilizing at high temperature, finally subpackaging in an aseptic culture device (generally an aseptic culture bottle/tube), and sealing with a high-temperature resistant sealing film;

s2, collecting fly eggs:

s21, preparing an egg collecting flat plate: mixing water, concentrated succus Vitis Viniferae and agar, boiling, packaging into sterile plate, and spreading appropriate amount of yeast powder after it is solidified;

s22, preparing a fruit fly egg collecting cup: reversely buckling a transparent plastic cup with the caliber slightly smaller than that of the plate on the egg collecting flat plate in the step S21, and pricking a hole at the bottom of the cup, wherein the diameter of the hole is smaller than that of the adult fruit fly;

s23, spawning: transferring the parent male and female drosophila melanogaster imagoes to the drosophila melanogaster egg collecting cup in the step S22, placing the egg collecting plate close to the lower part of the egg collecting plate in an incubator for culturing, and beginning to collect eggs after culturing for 4 hours;

s24, collecting and checking: carefully inverting the fruit fly egg collecting cup, slightly vibrating the fruit fly to the bottom of the cup, taking up the egg collecting flat plate, replacing the flat plate with a new flat plate, observing the egg collecting flat plate by using a microscope, throwing away hatched larvae, adding water into the flat plate, transferring the eggs to a cell filter screen, and collecting the fruit fly eggs;

s3, aseptic culture of drosophila eggs:

s31, sterilizing and subpackaging the egg surface in a superclean bench: placing the mesh screen containing the fly eggs in the step S24 in a culture dish, washing the eggs with 2.7% sodium hypochlorite solution for 2min, repeating twice, washing the eggs with sterilized distilled water, washing the eggs with 70% ethanol solution for 2min, repeating twice, and washing off residual ethanol on the surfaces of the eggs with sterilized distilled water; then subpackaging the rinsed eggs into a sterile culture medium, sealing the eggs with a sealing film, transferring the eggs into an incubator to be cultured with the ordinary fruit flies which are not disinfected and sterilized, and preliminarily judging whether the eggs are infected with bacteria according to the color of the culture medium, whether bubbles appear and the growth cycle of the fruit flies;

s4, sterile fruit fly model verification:

s41, coating plate verification: mashing the eclosion imagoes of the fruit flies and coating the eclosion imagoes on an LB solid culture medium, wherein aseptic colonies grow to obtain aseptic fruit flies;

PCR verification of S42 and 16S colonies: extracting the DNA of the adult drosophila melanogaster, carrying out 16S rDNAPCR amplification by using the bacterial universal primers shown in SEQ ID NO. 1 and SEQ ID NO. 2, and obtaining the sterile drosophila melanogaster without a band at 1464 bp.

Preferably, the drosophila include, but are not limited to, wild type W¹¹¹⁸The strain Drosophila melanogaster.

Preferably, the adult fruit fly at step S23 is a fruit fly after emergence for 2-5 days.

Preferably, in step S21, 50mL of concentrated grape juice, 3g of agar and 0.3g of yeast powder are added per 100mL of water.

Preferably, the pore size of the cell sieve is 100 μm.

Preferably, the culture conditions of step S23 are: the temperature was 25 ℃ and the humidity was 60%.

Preferably, the culture conditions of step S31 are: the temperature is 25 ℃, the humidity is 60%, and the ratio of light to dark is 12h to 12 h.

Preferably, the culture conditions of step S41 are: culturing at 37 deg.C for 24 h.

Preferably, the split in step S21 is performed in a clean bench, and the split is started when the temperature of the culture medium is reduced to 55-65 ℃.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a preparation method of aseptic fruit flies, which technically optimizes a plurality of aspects of a fruit fly egg collecting device, preparation of an aseptic culture medium, rinsing sterilization and aseptic culture of eggs, and the like, for example, a high-temperature sealing film commonly used in tissue culture technology is used for sealing a culture bottle/tube, can resist the high temperature of 135 ℃, can perform damp and hot sterilization, can ensure air exchange, and can achieve the aim of sealing and preventing bacteria. Generally, the method can create a sterile and breathable growth environment for the growth of the fruit flies, can ensure the normal growth of the fruit flies, can greatly reduce the probability of contamination in the culture process, has the advantages of simple and convenient method, easy operation, low cost, good repeatability, high success rate, large-scale preparation and the like, and has important application value in the research of the field of life science. In particular, the advantages of the invention are embodied in the following aspects:

(1) the requirement on the culture environment is low: in order to maintain the aseptic growth environment of the fruit flies, the fruit flies die greatly due to the fact that bacteria are completely sealed, the high-temperature resistant sealing film with the bacteria isolation effect and the ventilation effect is adopted, and the feeding difficulty of the aseptic fruit flies is greatly reduced. In addition, the sealing film can be matched with various fruit fly culture bottles and culture tubes, and has better compatibility. The sterile fruit fly culture bottle using the sealing film can be placed in an incubator together with common fruit flies for breeding, and the problem of bacterial contamination can be avoided as long as the sealing film is not damaged.

(2) The operation is simple and convenient: compared with other sterile animal models, the drosophila model is easy to culture, rapid in propagation and short in growth cycle, and is a classical biological research model. The method can be suitable for fruit flies of various strains and is used for removing extracellular microorganisms to construct a sterile fruit fly model. The whole operation flow is simple and easy to operate.

(3) No antibiotics were used: the traditional aseptic fruit fly preparation method uses antibiotics, but the antibiotics have certain toxicity, are easy to generate drug resistance, are difficult to completely remove most of microorganisms in fruit flies, and the like, and the antibiotics are various and inconvenient to select, are not high-temperature resistant, and need special solvents to dissolve in some cases.

(4) The cost is low: the reagent materials used in the whole experimental process are common living things in a biological laboratory, are convenient to purchase and low in cost, only need a conventional high-temperature sterilization pot and a clean bench, and have low requirements on laboratory equipment.

(5) The model has good repeatability and high success rate, and can be prepared in large scale: the preparation method of the aseptic fruit flies has strong stability and high success rate, and in addition, the culture quantity of the aseptic fruit flies can be enlarged by using the culture bottles so as to meet the requirements of experimental materials.

Drawings

FIG. 1 is a fruit fly egg collecting cup;

FIG. 2 shows a thread-drawing pen (A) and a screen (B);

FIG. 3 is a sterile culture flask;

FIG. 4 is a sterile culture tube;

FIG. 5 shows the results of plate-coating verification (A) and colony PCR verification (B) of aseptic fruit flies (C is common fruit flies and S is aseptic fruit flies);

FIG. 6 shows the effect of different yeast concentrations on the growth and development of Drosophila.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

Example 1A simple and efficient method for preparing aseptic Drosophila melanogaster

1. Preparation of sterile Medium

(1) Preparation of fruit fly culture medium: 50g of yeast, 100g of glucose, 12g of agar and 2g of potassium sorbate are weighed in a glass conical flask, 1L of distilled water is added to the mixture, the mixture is mixed uniformly, and the culture medium is heated and boiled in a microwave oven.

(2) High-temperature sterilization: the drosophila culture medium is put into a sterilization pot to be sterilized for 25 minutes at the high temperature of 121 ℃. In this procedure, an empty Drosophila culture flask/tube (sealed with a high temperature resistant sealing film), distilled water (200mL) and a crochet pen were sterilized at high temperature.

(3) Subpackaging a sterile culture medium: and after the sterilization is finished, taking out the culture medium, placing the culture medium in a super-clean workbench, opening the ultraviolet sterilization for 30 minutes, shaking the culture medium to fully mix the culture medium after the sterilization is finished, subpackaging the culture medium in sterile culture bottles/tubes when the temperature of the culture medium is reduced to about 60 ℃, deliberately shaking the culture medium to mix the culture medium in the cooling and solidification process, and sealing the culture medium with a sealing film again to prevent the contamination of bacteria.

2. Collecting fly eggs

(1) Preparing parent fruit flies: preparing in advance about 500 times of the wild type W after 2-5 days of emergence¹¹¹⁸Adult drosophila (purchased from the university of qinghua drosophila), male and female.

(2) Preparing an egg collecting plate: mixing 100mL of distilled water, 50mL of concentrated grape juice (GB/T31121, Zhejiang Denxin beverage Co., Ltd.) and 3g of agar, boiling in a microwave oven for 3 times, packaging into a clean plate (subjected to high temperature sterilization), cooling, solidifying, and spreading about 0.3g of yeast powder. Grape juice can attract fruit flies to stay on the plate for oviposition, the culture medium is changed into purple, white eggs laid by the fruit flies can be more easily identified by naked eyes, and in addition, the fruit flies can be promoted to lay eggs by ingesting yeast.

(3) Preparing a fruit fly egg collecting cup: and (3) reversely buckling a disposable transparent plastic cup (the caliber of the plate is 9cm, and the caliber of the plastic cup is 8cm) with the caliber slightly smaller than that of the plate on the egg collecting flat plate prepared in the step (2), pricking a through hole with the size of about 0.2mm at the bottom of the cup by using a heated needle for air circulation, wherein the diameter of the through hole is smaller than that of an adult fruit fly to prevent the fruit fly from drilling out, and the upper part of the egg collecting cup for the fruit fly is provided with the plastic cup and the lower part of the egg collecting cup for the fruit fly is provided with the plate as shown in figure 1.

(4) Spawning: transferring the parent male and female fruit fly imagoes to a fruit fly egg collecting cup prepared in advance after stunning with carbon dioxide, covering an egg collecting flat plate, and checking whether obvious gaps exist or not to prevent the fruit flies from flying away; after 5-8 minutes the flies recovered from coma, the egg collecting device was placed in an incubator at 25 ℃ and 60% humidity, carefully inverted (plate under, plastic cup over) and timed to begin collecting eggs after 4 hours of incubation.

(5) Collecting and checking: carefully inverting the fruit fly egg collecting cup (with the plate on top and the plastic cup on the bottom), holding the cup body by hand, gently shaking the fruit fly to the bottom of the cup, quickly taking up the egg collecting plate and replacing with a new plate. The egg receiving plate is placed under an inverted microscope and carefully examined for hatched larvae which must be picked out for disposal or which can lead to contamination of the sterile fruit flies. An appropriate amount of distilled water (5 mL/plate) was then added to the plate and the eggs were transferred to a cell screen of 100 μm pore size using a clean crochet pen to collect the Drosophila eggs (the crochet pen and screen are shown in FIG. 2). And (3) slightly washing the collected fruit fly eggs twice with distilled water, taking out a part of eggs, subpackaging the eggs into a sterile culture medium to serve as the common fruit flies of a control group, and using the rest eggs to prepare the sterile fruit flies.

3. Aseptic culture of Drosophila melanogaster eggs

(1) Preparing a rinsing liquid: 100mL of each of a 2.7% sodium hypochlorite solution (concentration by mass/volume) and a 70% ethanol solution (concentration by volume) was prepared using distilled water.

(2) Sterilization, disinfection and packaging of egg surfaces (operation in clean bench):

materials required: subpackaging the obtained product with sterile culture medium, sterilized thread drawing pen, sterilized distilled water (200mL), disposable sterile culture dish, 2.7% sodium hypochlorite solution, 70% ethanol solution, alcohol lamp, marking pen, etc.

Placing the filter screen containing the drosophila ova in a culture dish, washing the ova with 2.7% sodium hypochlorite solution for 2min, repeating for 2 times, and each time for 2 min; washing ovum with 70% ethanol solution for 2min, repeating for 2 times (2 min each time), and washing off residual ethanol on ovum surface with sterilized distilled water.

Thirdly, subpackaging the rinsed eggs into a sterile culture medium by using a line drawing pen, sealing the eggs by using a sealing film, and transferring the eggs into an incubator (a sterile culture bottle and a sterile culture tube are respectively shown in figures 3 and 4) to be cultured together with the common drosophila melanogaster (the drosophila melanogaster eggs which are not sterilized) of a control group, wherein the culture temperature is 25 ℃, the humidity is 60%, and the ratio of illumination to darkness is 12h to 12h (namely, the light and darkness are alternated for 12h respectively).

And fourthly, the growth condition of the fruit flies is observed regularly, compared with the normal germ-bearing fruit flies, the growth cycle of the germ-free fruit flies is obviously delayed, and the pupation time is delayed for about 2 to 3 days. The color of the sterile culture medium is brownish yellow, and is darker than that of the culture medium of the control group, and the sterile culture medium has no bubbles, so that whether the bacteria are infected can be preliminarily judged according to the color of the culture medium, whether the bubbles appear and the growth period of the drosophila.

4. Sterile fruit fly model verification

(1) Plate coating verification: adults (cultured for about 14 days) 2-3 days after the emergence of the sterile fruit flies were mashed and spread on LB solid medium, and cultured at 37 ℃ for 24h (with the control group of the common fruit flies as a control), the control group of the common fruit flies had a large number of colonies growing, and the sterile fruit flies had no colonies growing, as shown in FIG. 5A.

(2)16S colony PCR verification: adult Drosophila DNA (sterile Drosophila and control group of common Drosophila) was extracted and 16S rDNA PCR amplification was performed using bacterial universal primers [ 27F:5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO:1), 1492R:5'-taccttgttacgactt-3' (SEQ ID NO:2) ]. And (3) PCR system: 2 XTaq PCR mix (Quikcloning) 10. mu.l, upstream and downstream primers 27F and 1492R 0.5. mu.l each, DNA template 2. mu.l (50-100ng), sterile water 7. mu.l. And (3) PCR reaction conditions: 94 ℃ for 2 min; circulating for 30 times at 94 deg.C for 30sec, 60 deg.C for 30sec, and 72 deg.C for 1.5 min; 5min at 72 ℃. The result of 1% agarose gel electrophoresis shows that the common fruit fly has a distinct band at 1464bp, while the sterile fruit fly has no band at the corresponding position, as shown in FIG. 5B.

According to statistics, the success rate of the method for preparing the sterile fruit flies is up to 99%, and the success rate of the method for preparing the sterile fruit flies is basically consistent after three repeated cultures, which shows that the method for preparing the sterile fruit flies has high success rate and good repeatability.

EXAMPLE 2 optimization of the preparation method of sterile Drosophila

Before the preparation of the sterile fruit flies in the example 1, the preparation method of the sterile fruit flies is optimized, and the specific optimization items are as follows:

(1) optimizing an egg disinfection process: the sodium hypochlorite can remove chorions on the surfaces of the drosophila ova, the sterilization and disinfection effects are in direct proportion to time within a certain range, and meanwhile, the sodium hypochlorite has certain toxicity, so 2.7% sodium hypochlorite solution is adopted to wash the drosophila ova for 1min, 2min and 5min respectively twice, and the result shows that microorganisms on the surfaces of the ova cannot be completely removed in 1 min; when the incubation time is 5min, the incubation rate is too low, and the obtained quantity of the adult drosophila melanogaster cannot meet the requirements of the next experiment; therefore 2min sterilization was chosen twice.

(2) Optimization of the sealing mode of the sterile fruit fly culture device: to ensure that the sterile fly eggs grow in a sterile medium environment and remain sterile for long periods of time, a total of three sealing schemes have been attempted: the culture bottle is sealed by a closed culture bottle, an anti-mite plug and a high-temperature-resistant sealing film. The result shows that the fruit flies can not grow normally due to complete sealing; the anti-mite plug has small pore diameter to block part of bacteria, but the contamination probability is obviously increased after high-temperature sterilization; and the high-temperature resistant sealing film well meets the requirement of bacteria isolation, so that the sealing mode of using the high-temperature resistant sealing film as the sterile fruit fly culture device is determined.

(3) Optimization of a sterile fruit fly culture medium: different culture mediums have great influence on the growth and development of the fruit flies, in order to eliminate the influence of various factors, a yeast-glucose culture medium (yeast, glucose, agar and potassium sorbate) with simple components is selected, the content of the yeast is set to be 2.5 percent of yeast, 5 percent of yeast and 10 percent of yeast (10 percent of glucose, 1.2 percent of agar and 0.2 percent of potassium sorbate), and the influence of different yeast concentrations on the growth and development of the fruit flies is compared (the aseptic fruit flies and the common fruit flies of a control group are cultured simultaneously).

The experimental result is shown in fig. 6, when the yeast content is 2.5%, the control group of the common drosophila pupate around day 11, but the aseptic drosophila can not pupate normally, and all the drosophila pupate die in the larval stage; when the yeast content is 5%, the aseptic fruit flies pupate about 3 days later than the common fruit flies in the control group, namely the existence of flora can obviously influence the development cycle of the fruit flies; when the yeast content is 10%, the pupation time of normal drosophila and aseptic drosophila in the control group is not obviously different due to the over-rich nutrition of the culture medium, and the influence of the flora on the developmental cycle of drosophila is not obvious. This result demonstrates that nutritional conditions can modulate the impact of the flora on the host developmental cycle. Therefore, the 5% yeast concentration was chosen to allow normal survival of the sterile fruit flies, while significant changes in the developmental cycle of the flies due to sterility were observed.

(4) And (3) verifying a sterile fruit fly model: the traditional aseptic fruit fly model verification method generally only adopts a single plate coating method to verify whether fruit flies are infected with bacteria or not, and although most of intestinal flora are anaerobic bacteria and can not grow on a solid LB culture medium, the false positive phenomenon still exists only by the plate coating verification. And whether the drosophila imagoes carry bacterial colonies is verified by adopting 16S bacterial colony PCR through a molecular means, and the bacterial nucleic acid can be specifically amplified through a 16S rDNA primer as long as the extracted total DNA contains the bacterial nucleic acid, so that whether the drosophila imagoes are infected with bacteria or not is detected. Therefore, the aseptic fruit fly model is verified simultaneously by adopting two methods of plate coating on aseptic fruit fly adults and 16S colony PCR, and the verification result is more credible by combining the two methods.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Sequence listing

<110> university of south China

<120> simple, convenient and efficient preparation method of aseptic fruit flies

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 27F

<400> 1

agagtttgat cctggctcag 20

<210> 2

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 1492R

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taccttgtta cgactt 16

Claims

1. A simple, convenient and efficient preparation method of aseptic fruit flies is characterized by comprising the following steps:

s1, preparing a sterile culture medium: weighing 5% of yeast, 10% of glucose, 1.2% of agar and 0.2% of potassium sorbate, adding water, mixing uniformly, heating and boiling, then performing high-temperature sterilization, finally subpackaging in an aseptic culture device, and sealing with a high-temperature resistant sealing film;

s2, collecting fly eggs:

s3, aseptic culture of drosophila eggs:

s4, sterile fruit fly model verification:

2. A simple and efficient bag-free as defined in claim 1Preparation method of bactrocera dorsalis, characterized in that the bactrocera dorsalis includes but is not limited to wild type W¹¹¹⁸The strain Drosophila melanogaster.

3. The method for preparing sterile fruit flies in simple and efficient manner according to claim 1, wherein the adult fruit flies are fruit flies which have emerged for 2-5 days in step S23.

4. The method of claim 1, wherein 50mL of concentrated grape juice, 3g of agar and 0.3g of yeast powder are added to 100mL of water in step S21.

5. The method as claimed in claim 1, wherein the cell filter screen has a pore size of 100 μm.

6. The method for preparing sterile fruit flies according to claim 1, wherein the culture conditions in step S23 are as follows: the temperature was 25 ℃ and the humidity was 60%.

7. The method for preparing sterile fruit flies according to claim 1, wherein the culture conditions in step S31 are as follows: the temperature is 25 ℃, the humidity is 60%, and the ratio of light to dark is 12h to 12 h.

8. The method for preparing sterile fruit flies according to claim 1, wherein the culture conditions in step S41 are as follows: culturing at 37 deg.C for 24 h.

9. The method for preparing sterile fruit flies in simple and efficient manner as claimed in claim 1, wherein the dispensing in step S21 is performed in a clean bench, and the dispensing is started when the temperature of the culture medium is lowered to 55-65 ℃.