Aseptic culture method for corpse flies

Abstract

The invention discloses a sterile culture method for corpse flies. The sterile culture method comprises the following steps: step 1, treating primary chrysomyia megacephala larvae; step 2, preparing a sterile feed; and 3, performing sterile culture on the chrysomyia megacephala. Compared with the prior art, the method disclosed by the invention has the advantages that (1) the corptrophile flies in a sterile culture system are not interfered by external microorganisms, so that a researcher can more accurately control experimental conditions, and the interaction between the corptrophile flies and the microorganisms is more accurately researched; for example, how the microorganisms influence the growth, development, metabolism, behaviors and the like of the corpse flies; and (2) through a sterile culture system, the corptrophile fly microbiome can be accurately controlled, so that how the symbiotic flora affects various characters of the host and the influence of different flora on corptrophile insects at different stages are clarified, such as host metabolism, insecticide resistance, host immunity and the like.

Description

Technical Field

[0001] The invention relates to the technical field of culturing sarcophagous flies, and in particular to a sterile culturing method for sarcophagous flies. Background Art

[0002] The estimation of postmortem interval (PMI) is one of the key links in solving criminal cases. Currently in the field of forensic medicine, sarcophagous flies play an important role in estimating the PMI of decaying corpses. Through research, a series of growth and development data of sarcophagous flies have been mastered, and standard methods for collecting and breeding sarcophagous flies in the laboratory have been developed. The growth and development data of sarcophagous flies cultured in the laboratory play a vital role in estimating the PMI of decaying corpses.

[0003] However, when we applied the growth and development data of sarcophagous flies collected in the above-mentioned conventional laboratories to PMI inference, we found that there was a large deviation between it and the actual time of death. At the same time, many studies have suggested that the underlying reason for the differences in development and behavior of flies in laboratories and natural environments is the difference in their intestinal microorganisms. Studying the effects of intestinal microorganisms on the growth and development of sarcophagous flies can provide support for the establishment of a more accurate PMI system at the microbial level. However, in conventional (bacterial) laboratory environments, it is difficult to experimentally study the mechanism of the effect of a certain intestinal microorganism on the growth and development of sarcophagous flies.

[0004] Therefore, in order to better study the mechanism by which intestinal microorganisms affect the growth and development of sarcophagous flies, a sterile culture method for sarcophagous flies was constructed for the first time. During the culture process of this culture method, the growth and development of sarcophagous flies will not be disturbed by the microorganisms in the external environment. We can control variables, such as adding different antibiotics or different intestinal microorganisms during the culture process, and obtain the growth and development parameters of sarcophagous flies under these variables through various experiments. These growth and development parameters are compared with those of sarcophagous flies under conventional laboratory culture conditions. At the same time, a series of experimental studies are carried out to explore the impact of intestinal microorganisms on the growth and development of sarcophagous flies, providing a basis for building a more accurate PMI inference system. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for aseptic cultivation of sarcophagous flies in view of the deficiencies raised in the above-mentioned background technology.

[0006] In order to solve the above technical problems, the present invention provides a technical solution: a method for aseptically culturing sarcophagous flies, which comprises the following steps:

[0007] Step 1: Treatment of the first generation of golden fly larvae;

[0008] Step 2: Preparation of sterile feed;

[0009] Step 3: Aseptic culture of Chrysopa longituba.

[0010] Furthermore, in step 1, the eggs that have just been laid by the big-headed golden fly are selected, and the surface of the eggs is first rinsed with clean water for 3 times, and then the eggs are placed in 75% disinfectant alcohol for disinfection for 3 minutes, and then the disinfected eggs are rinsed with pure water for 3 times to wash away the alcohol, and the rinsed eggs are immersed in 0.05% sodium hypochlorite solution for sterilization for 3 minutes.

[0011] Furthermore, the sterilized eggs were finally rinsed three times with PBS buffer.

[0012] Furthermore, in the step 2, 85g of bran and 15g of fish meal are fully mixed and placed in a tin foil box, which is then sealed. The tin foil box is then placed in an aluminum box and the lid is closed. 1000ml of clean water is put into a 1000ml canning bottle, the bottle mouth is slightly loosened, and the bottle is sterilized in a high-pressure steam sterilizer at 0.11MPa and 121°C for 30min, and cooled for standby use.

[0013] Furthermore, the sterile bran feed and clean water prepared in step 2 are transferred into a sterile isolator and sterilized using an ultraviolet lamp for 30 minutes.

[0014] Furthermore, in step three, a plurality of cylindrical plastic insect raising boxes with a diameter of about 10 cm and a height of about 8 cm are selected and sterilized, and then placed in a sterile isolation bag; the sterilized sterile feed is mixed with clean water in the insect raising box in a ratio of about 1:2 to create a moist sterile culture environment; 1 g of sterilized golden fly eggs that have been disinfected and sterilized are placed in one of the sterile plastic insect raising boxes containing sterile feed that has just been prepared; the remaining feed and plastic insect raising boxes are kept for later use.

[0015] Furthermore, after the big-headed golden fly is cultured to pupation, the pupae are picked out from the feed and transferred to a new plastic insect box, and then the plastic insect box is transferred to a 35cm*35cm*35cm insect cage, and the pupae are continued to be raised until they emerge and lay eggs, completing a culture cycle, and the feed is changed every other day during the culture period;

[0016] After adopting the above structure, the present invention has the following advantages: (1) There is no interference from external microorganisms in the sarcophagous flies under the sterile culture system, so that researchers can control the experimental conditions more accurately, thereby more accurately studying the interaction between sarcophagous flies and microorganisms, such as how microorganisms affect the growth, development, metabolism and behavior of sarcophagous flies.

[0017] (2) Through the sterile culture system, the microbiome of sarcophagous flies can be precisely manipulated to elucidate how the symbiotic microbiota affects various traits of the host, as well as the effects of different microbiota on sarcophagous insects at different stages, such as host metabolism, insecticide resistance, and host immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the culture medium results in an isolation bag of a sterile culture method for sarcophagous flies of the present invention.

[0019] Figure 2 It is a schematic diagram of the culture medium results outside the isolation bag of the aseptic culture method of sarcophagous flies of the present invention.

[0020] Figure 3 It is a schematic diagram of the environmental sterility detection results of the aseptic culture method of sarcophagous flies of the present invention.

[0021] Figure 4 It is a schematic diagram of the sterility detection results of the Chrysopa larvae of the present invention in the sterile culture method of sarcophagous flies.

[0022] Figure 5 It is a schematic diagram of the body length development curve of the big-headed golden fly under different culture conditions of a sterile culture method of sarcophagous flies of the present invention. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0024] A method for aseptically culturing sarcophagous flies comprises the following steps:

[0025] Step 1: Treatment of primary generation of big-headed golden fly larvae: In the step 1, newly laid eggs of big-headed golden fly are selected, and the surface thereof is first rinsed 3 times with clean water, and then the eggs are disinfected in 75% disinfectant alcohol for 3 minutes, and then the disinfected eggs are rinsed 3 times with pure water to wash away the alcohol, and the rinsed eggs are immersed in 0.05% sodium hypochlorite solution for sterilization for 3 minutes, and finally the sterilized eggs are rinsed 3 times with PBS buffer.

[0026] Step 2: Preparation of sterile feed: In the step 2, 85g of bran and 15g of fish meal are fully mixed and placed in a tin foil box, which is then sealed. The tin foil box is then placed in an aluminum box and the lid is closed. 1000ml of clean water is put into a 1000ml canned bottle, the bottle mouth is slightly loosened, and the bottle is sterilized in a high-pressure steam sterilizer at 0.11MPa and 121°C for 30min, cooled for standby use, and the sterile bran feed and clean water prepared in step 2 are moved into a sterile isolator, and then sterilized using an ultraviolet lamp for 30min.

[0027] Step 3: Aseptic culture of big-headed golden fly: In the step 3, multiple cylindrical plastic insect boxes with a diameter of about 10 cm and a height of about 8 cm are selected and sterilized, and then placed in a sterile isolation bag; the sterilized sterile feed is mixed with clean water in the insect box in a ratio of about 1:2 to create a moist sterile culture environment; 1 g of sterilized big-headed golden fly sterile eggs that have been disinfected and sterilized are placed in a sterile plastic insect box containing sterile feed that has just been prepared; the remaining feed and plastic insect box are kept aside; after the big-headed golden fly is cultured to pupation, the pupae are picked out from the feed and transferred to a new plastic insect box; the plastic insect box is then transferred to a 35 cm*35 cm*35 cm insect cage; the pupae are continued to be raised until they emerge and lay eggs, completing a culture cycle; the feed is changed every other day during the culture period.

[0028] During the cultivation process, in order to ensure that the cultivation process is not interfered by external microorganisms, the following experimental verifications were set up:

[0029] (1) Air sterility test:

[0030] Purpose: To test whether there are microorganisms in the culture process in the sterile isolation package, and to verify whether the culture process is interfered by external microorganisms;

[0031] Specific operation: During the culture process, four culture media, TSA, nutrient agar, BHI and blood agar, are placed inside and outside the isolation bag respectively, and the results are observed after the culture process is completed;

[0032] like Figure 1 , 2 As shown, the result: Figure 1 , 2 The right side shows that no colonies are generated in the four culture media in the sterile isolation package. Compared with the outside of the sterile isolation package, the sterile state in the sterile isolation package during the culture process can be verified;

[0033] Figure 1 : Culture medium results in the isolation bag;

[0034] Figure 2 : Results of culture medium outside the isolation package.

[0035] (2) Environmental sterility testing:

[0036] Objective: To examine whether there are microorganisms in the environment during the growth and development of Chrysopa longituba;

[0037] Specific operation: randomly sample 3 places in the sterile isolator with a cotton swab soaked in physiological saline, and randomly sample 3 places in the food. After nucleic acid extraction and purification using a bacterial DNA kit, PCR amplification is performed using universal true bacterial primers. At the same time, the same method is used to sample food in a conventional (bacterial) laboratory environment as a positive control group to observe the results;

[0038] like Figure 3 Shown: Environmental sterility test results: AC, positive control; SP, space sampling test results inside the isolation bag; Food, food sampling test results.

[0039] (3) Aseptic larvae detection: Take the big-headed golden fly larvae in the sterile isolator and put them into a centrifuge tube containing sterile PBS and glass sand (repeat twice), grind and homogenize, use a bacterial DNA kit to extract and purify the nucleic acid, and then use universal true bacterial primers for PCR amplification. At the same time, use the same method as above to sample larvae in a conventional (bacterial) laboratory environment as a positive control group to observe the results;

[0040] like Figure 4 Shown: Sterility test results of Chrysopa larvae: AC, positive control; Larva isolation bag larva sampling test results;

[0041] Under this sterile culture method, the big-headed golden fly completed a complete developmental period from egg to adult, indicating that this cadaveric fly can develop normally under this sterile culture method and can be used for scientific research. The following is the growth and development data of the big-headed golden fly under the sterile culture method and in a conventional laboratory environment.

[0042] (4) Comparison of the growth and development curve of the big-headed golden fly under the sterile culture method and the growth and development curve of the big-headed golden fly in the conventional laboratory environment: Figure 5 Shown: the body length development curve of the big-headed golden fly under different culture conditions (the indoor wheat bran + fish meal group and the 25°C wheat bran + fish meal group are the growth and development data of the big-headed golden fly in a conventional laboratory environment, and the sterile group is the growth and development data under the sterile culture method in a constant temperature room at 25°C).

[0043]

[0044] Table 1: Mean development time of different life stages of Chrysocytidae (Mean±SD)

[0045] The present invention and its implementation methods are described above, and such description is not restrictive, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by it, and does not deviate from the purpose of the invention, and does not creatively design a structure and implementation method similar to the technical solution, they should all fall within the protection scope of the present invention.

Claims

1. A method for aseptic cultivation of sarcophagous flies, characterized in that: It includes the following steps: Step 1: Treatment of the first generation of golden fly larvae; Step 2: Preparation of sterile feed; Step 3: Aseptic culture of Chrysopa longituba.

2. The method for aseptic cultivation of sarcophagous flies according to claim 1, characterized in that: In the step 1, newly laid eggs of the big-headed golden fly are selected, and the surface of the eggs is first rinsed with clean water for 3 times, and then the eggs are placed in 75% disinfectant alcohol for disinfection for 3 minutes, and then the disinfected eggs are rinsed with pure water for 3 times to wash away the alcohol, and the rinsed eggs are immersed in 0.05% sodium hypochlorite solution for sterilization for 3 minutes.

3. The method for aseptic cultivation of sarcophagous flies according to claim 2, characterized in that: Finally, the sterilized eggs were rinsed three times with PBS buffer.

4. The method for aseptic cultivation of sarcophagous flies according to claim 1, characterized in that: In the step 2, 85g of bran and 15g of fish meal are mixed thoroughly and placed in a tin foil box, which is then sealed. The tin foil box is then placed in an aluminum box and the lid is closed. 1000ml of clean water is put into a 1000ml canning bottle, the bottle mouth is slightly loosened, and the bottle is sterilized in a high-pressure steam sterilizer at 0.11MPa and 121°C for 30min, and cooled for later use.

5. The method for aseptic cultivation of sarcophagous flies according to claim 4, characterized in that: Transfer the sterile bran feed and clean water prepared in step 2 into a sterile isolator, and sterilize using an ultraviolet lamp for 30 minutes.

6. The method for aseptic cultivation of sarcophagous flies according to claim 1, characterized in that: In the step three, a plurality of cylindrical plastic insect raising boxes with a diameter of about 10 cm and a height of about 8 cm are selected and sterilized, and then placed in a sterile isolation bag; the sterilized sterile feed is mixed with clean water in a ratio of about 1:2 in the insect raising box to create a moist sterile culture environment; 1 g of sterilized golden fly eggs are placed in one of the sterile plastic insect raising boxes containing sterile feed that has just been prepared; the remaining feed and plastic insect raising boxes are kept aside for later use.

7. The method for aseptic cultivation of sarcophagous flies according to claim 6, characterized in that: After the big-headed golden fly is cultured to pupation, pick out the pupae from the feed and transfer them to a new plastic insect box. Then transfer the plastic insect box to a 35cm*35cm*35cm insect cage. Continue to raise them until they emerge and lay eggs, completing a culture cycle. During the culture period, change the feed every other day.

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