A culture medium for culturing Exorista sorbillans in vitro and its culturing method

Abstract

The present invention provides a culture medium for in vitro culturing Exorista sorbillans Wiedemann and a culturing method thereof. The culture medium includes a liquid culture medium and sterile absorbent cotton. The liquid culture medium is a mixed solution of skim milk, sucrose, yeast extract, egg yolk, silkworm hemolymph, and gentamicin sulfate. The culturing method of the present invention mainly includes attracting Exorista sorbillans Wiedemann to lay eggs with live insects at a certain temperature, and culturing Exorista sorbillans Wiedemann larvae in vitro with an optimized artificial culture medium. The present invention uses live insects to collect a large number of Exorista sorbillans Wiedemann eggs, overcoming the problem of difficult acquisition of fly eggs under the condition of no host. At the same time, adding silkworm hemolymph at a specific period to the in vitro culture medium significantly reduces the development time of maggots and improves the adult fly rate of Exorista sorbillans Wiedemann. The parasitic effect of the in vitro cultured tachinid fly obtained by the present invention has no obvious difference from that of the adult fly parasitized in the host. The above materials are widely available, with low costs, and the operation is simple and feasible, and the whole-year culturing and subculture of Exorista sorbillans Wiedemann can be completely realized.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a culture medium for culturing Exorista sorbillans Wiedemann in vitro and a culturing method thereof. Technical Background

[0002] Exorista sorbillans Wiedemann belongs to the family Tachinidae and the genus Exorista, and is a large-egg tachinid fly. It can parasitize various Lepidoptera insects and agricultural and forestry pests including silkworms. At present, the control of Exorista sorbillans Wiedemann in China mainly relies on the silkworm medicine "Miecanying" developed in the 1960s. Due to the long-term use of a single drug, it has been reported that fly maggots in some silkworm areas have developed drug resistance. At this time, the identification and analysis of drug resistance genes and genes related to growth and development of Exorista sorbillans Wiedemann are particularly important. However, in the natural state, Exorista sorbillans Wiedemann can only reproduce and develop in summer and autumn. This tachinid fly parasitizes internally, making it impossible to observe the morphological development of tachinid fly larvae and directly conduct experimental treatments during the larval development process. Therefore, developing a method for culturing Exorista sorbillans Wiedemann in vitro can greatly promote the experimental research on Exorista sorbillans Wiedemann.

[0003] The natural reproduction of Exorista sorbillans Wiedemann is by laying eggs on the body surface of silkworms or other insects. After the larvae hatch and enter the host body, the tail is fixed to the silkworm epidermis to form a "respiratory funnel" communicating with the outside world to ensure respiration, while the larval trunk and head are in the silkworm body cavity, mainly feeding on the silkworm hemolymph and fat body. Since silkworms are its main host, the growth and reproduction of Exorista sorbillans Wiedemann are often restricted by seasons.

[0004] Most studies on artificial culture media for the family Parasitic flies use beef extract, insect cell culture media, host homogenate and a large amount of host hemolymph as ingredients, which consume a lot and are costly. In recent years, egg yolk has been used as the main nutrient for the development of fly maggots, reducing costs while obtaining a certain proportion of adult flies. However, promoting the normal reproduction and transmission of parasitic flies is inseparable from host tissue nutrients. The earliest culture of silkworm parasitic flies used beef broth, egg yolk, and skim milk as the main nutrients, and 0.5% agar as the supporting material. The obtained adult fly pupae had low weight and short lifespan. After adding 13.2% silkworm hemolymph, the adult fly rate was only 5.7%, and the larval stage was longer than that in the natural parasitic state. The addition of agar can cause swelling of the fly maggot intestine, inhibit intestinal absorption, and affect the adult fly rate. Japan used wheat bran, soybean flour, yeast, sucrose, and toilet paper to prepare the supporting material and add the fifth-instar fourth-day silkworm hemolymph, with an adult fly rate of 4-8%. The silkworm hemolymph consumption was large and the adult fly rate was low. Existing studies have pointed out that the amino acid composition in the culture medium has a great influence on the growth of parasitic flies. Plant proteins such as wheat bran and soybean meal may not be suitable for the food needs of parasitic flies, resulting in a high demand for silkworm hemolymph. Culture medium support materials At present, the in vitro culture method of gypsy moth parasitic flies has been simplified to remove beef extract, with only egg yolk and skim milk as the main nutrients and good results have been achieved, but the problem of the development time of fly maggots has not been solved. In addition, there has been no research on the problem of subculture of silkworm parasitic flies without a host. Summary of the invention

[0005] Purpose of the invention: In view of the defects and shortcomings of the prior art, the present invention provides a culture medium and method for attracting parasitic flies to lay eggs in a concentrated manner and to rapidly cultivate silkworm parasitic flies in vitro, so as to solve the current problems of difficulty in collecting eggs when silkworm parasitic flies have no host, high cost of in vitro culture, long development time of maggots, low adult fly rate, and inability to cultivate silkworm parasitic flies all year round (especially in winter) and pass on generations.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a culture medium for culturing silkworm parasitoids in vitro, the culture medium comprising a liquid culture medium and absorbent cotton, the liquid culture medium comprising skim milk, sucrose, yeast extract, egg yolk, hemolymph of silkworms (Jingsong×Haoyue five-instar silkworms) and a mixture of gentamicin sulfate. The mass volume ratio of the liquid culture medium to the absorbent cotton is 400μl:14-16mg.

[0007] Preferably, the mass volume ratio of the liquid culture medium to the absorbent cotton is 400 μl:15 mg.

[0008] The method for preparing the culture medium for in vitro culturing of silkworm parasitoid flies is specifically as follows:

[0009] (1) First, mix the skim milk and sucrose, then dissolve the yeast extract in sterilized water. After separately preparing them, sterilize them at high temperature, let them cool, and then mix them. Then, successively add egg yolk, silkworm hemolymph, and gentamicin sulfate and mix well to obtain a liquid medium.

[0010] (2) Add the liquid medium prepared in step (1) to the sterilized absorbent cotton, spread it evenly, and then the medium for in vitro culturing Exorista sorbillans Wiedemann is obtained.

[0011] Among them, the silkworm hemolymph is extracted from the hemolymph of Jing Song × Hao Yue on the fourth day of the fifth instar (L5D4), the sixth day of the fifth instar (L5D6), or 2 weeks after mounting (2D).

[0012] Preferably, the silkworm hemolymph is extracted from Jing Song × Hao Yue on the sixth day of the fifth instar (L5D6).

[0013] Among them, the silkworm hemolymph is obtained by rinsing the silkworms of Jing Song × Hao Yue on the fourth day of the fifth instar (L5D4), the sixth day of the fifth instar (L5D6), or 2 weeks after mounting (2D) with pure water, heating them in a water bath at 60 °C for 10 min, wiping the silkworm bodies with 75% alcohol cotton balls under sterile conditions, volatilizing the alcohol, and then cutting the first pair of abdominal legs with a clean scissors to collect the hemolymph into a 1.5 ml EP tube and storing it at -20 °C. Take it out and melt it when in use.

[0014] Among them, when preparing 50 - 60 mL of the said medium, the composition ratio of each component is as follows: skim milk 20 - 40 mL; sucrose 0.8 - 1 g; yeast extract 2.5 - 3 g; egg yolk 5 - 6 ml; silkworm hemolymph 5 - 6 ml; gentamicin sulfate 90 - 120 μL, and the rest is sterile water.

[0015] Preferably, when preparing 54.4 mL of the said medium, the composition ratio of each component is as follows: skim milk 30 mL; sucrose 0.9 g; yeast extract 2.7 g; egg yolk 5.4 mL; silkworm hemolymph 5.5 mL; gentamicin sulfate 108.8 μL, and the rest is sterile water.

[0016] Preferably, the addition amount of silkworm hemolymph is 10% of the total volume of the liquid medium. Among them, the working concentration of the gentamicin sulfate is 8 - 9 μg / ml.

[0017] Preferably, the preparation method of the liquid medium is as follows: The medium is prepared by mixing two liquids, A and B. Liquid A is a mixed solution of skim milk and sucrose, and liquid B is a solution of yeast extract dissolved in sterilized water. Liquids A and B are separately prepared and then sterilized in an autoclave at 115 °C for 10 min (clumping will occur when milk and yeast extract are mixed and sterilized). After cooling, they are mixed, and egg yolk, silkworm hemolymph, and gentamicin sulfate are successively added to the mixed solution.

[0018] The method for culturing Exorista sorbillans in vitro using the culture medium described in the present invention is specifically as follows:

[0019] (1) Collect and process fly eggs: Use a species that attracts Exorista sorbillans to lay eggs to attract Exorista sorbillans to lay eggs. After laying eggs, remove the fly eggs.

[0020] (2) Place the fly eggs collected in step (1) in the culture medium for fly egg hatching and cultivation, and cultivate the maggots until they pupate.

[0021] (3) Collect and process fly pupae: Cultivate the fly pupae obtained in step (2) in complete darkness, collect the emerged tachinids for feeding, and these adult flies can be used for the next generation of parasitism.

[0022] Among them, the species that attracts Exorista sorbillans to lay eggs in step (1) is any one of the fifth-instar silkworms, silkworm pupae, Tenebrio molitor larvae, Antheraea pernyi pupae, and mature larvae of Exorista sorbillans.

[0023] Preferably, the species that attracts Exorista sorbillans to lay eggs is Antheraea pernyi pupae.

[0024] Among them, the egg-laying temperature of Exorista sorbillans in step (1) is 28 - 30 °C. In the present invention, when the temperature is increased to 28 °C in winter, the egg-laying amount increases significantly, while very few eggs are laid when the temperature is set at 25 °C.

[0025] Preferably, the egg-laying temperature of Exorista sorbillans in step (1) is 28 °C, and after the collection of fly eggs is completed, the temperature is adjusted back to 26 °C.

[0026] Among them, the method for taking fly eggs in step (1) is to soak the egg-laying carrier in clear water and gently rub it to collect the fly eggs, or gently pick the surface fly eggs with tweezers.

[0027] Preferably, the above-mentioned egg-taking time is within 48 h after the tachinid lays eggs; the egg-taking method is to soak the egg-laying carrier in clear water for 10 s, wear gloves and gently rub it to collect a large number of fly eggs, and use tweezers to gently pick the surface fly eggs for the above-mentioned other species.

[0028] Preferably, the specific cultivation method is as follows:

[0029] Put the fifth-instar silkworms, silkworm pupae, Antheraea pernyi pupae, Tenebrio molitor larvae, and mature larvae of Exorista sorbillans into the fly cage. Use tweezers to collect the fly eggs within 48 h after the tachinid lays eggs, disinfect them, rinse them twice with sterile water, then put the fly eggs into the prepared culture medium for fly egg hatching and cultivation. After pupation, take out the fly pupae and collect them uniformly in a clean petri dish. After emergence, put them into the fly cage and raise the adult flies indoors.

[0030] Among them, the selection of the species that attracts Exorista sorbillans to lay eggs: During the silkworm-raising period, the fifth-instar silkworms or silkworm pupae can be selected. During the non-silkworm-raising period, Tenebrio molitor larvae and Antheraea pernyi pupae, or mature larvae of Exorista sorbillans can be purchased from the market. Among them, the optimal one is Antheraea pernyi pupae.

[0031] Among them, the collection and treatment of the fly eggs: within 4 - 7 days after the emergence of Exorista sorbillans Wiedemann, place the egg - laying carrier into the fly cage, ensure the temperature at 28°C during the egg - laying period, collect the fly eggs within 48 hours after Exorista sorbillans Wiedemann lays eggs, disinfect them with 70% ethanol for 5 minutes, and rinse twice with sterile water.

[0032] Among them, the primary fly species of Exorista sorbillans Wiedemann: collect fly pupae in the silkworm room, and put them into the fly cage (length × width × height, 50 cm × 40 cm × 60 cm) after emergence, keep the temperature at 26 ± 1°C indoors, and raise them with 10% honey water.

[0033] Furthermore, use the fifth - instar silkworms, silkworm pupae, Tenebrio molitor, Antheraea pernyi pupae, and mature larvae of Exorista sorbillans Wiedemann to attract Exorista sorbillans Wiedemann to lay eggs. Remove the fly eggs within two days after egg - laying, disinfect them with 70% ethanol for 5 minutes, rinse twice with sterile water, then place the fly eggs in the culture medium, culture the maggots at 26°C under 0L:24D conditions until pupation, collect the fly pupae in a dry petri dish, place them in an incubator at 26°C under 0L:24D conditions for 12 days, collect the emerged flies in the fly cage, keep the temperature at 26°C under 12L:12D conditions, and feed them with 10% honey water every day. These adult flies can be used for the next experiment on parasitism.

[0034] The species attracting Exorista sorbillans Wiedemann to lay eggs mentioned above are the fifth - instar silkworms, silkworm pupae, Tenebrio molitor larvae, Antheraea pernyi pupae, and mature larvae of Exorista sorbillans Wiedemann. Among them, the optimal choice in winter is Antheraea pernyi pupae. The egg - laying temperature of the above - mentioned flies is 28°C, and the egg - taking time is within 48 hours after the flies lay eggs; the egg - taking method is to soak the egg - laying carrier in clear water for 10 seconds, gently rub it with gloves to collect a large number of fly eggs, or gently pick the surface fly eggs with forceps. The culture medium adds 10% of the total volume of the liquid culture medium of silkworm hemolymph from the fourth day of the fifth instar, the sixth day of the fifth instar, or 2 weeks after mounting of the silkworms, and preferably the silkworm hemolymph on the sixth day of the fifth instar.

[0035] The present invention provides a culture medium and method for completing the in - vitro culture of multiple generations of Exorista sorbillans Wiedemann. Using living insects to attract Exorista sorbillans Wiedemann to lay eggs, a large number of fly eggs are collected intensively under the condition of no host by regulating the temperature (the temperature for attracting flies to lay eggs is 28°C), which overcomes the problem of difficult acquisition of fly eggs under the condition of no host. In addition, adding silkworm hemolymph at a specific period to the in - vitro culture medium significantly reduces the development time of maggots and significantly increases the adult fly rate at the same time. The parasitism effect of the flies obtained by the present invention has no significant difference from natural parasitism. The above - mentioned materials are widely available, with low cost, and the operation is simple and feasible, and the cultivation and passage of Exorista sorbillans Wiedemann can be fully realized throughout the year (especially in winter).

[0036] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0037] (1) All the supplies used in the present invention are convenient to purchase and low in price. In particular, tussah silkworm pupae are in diapause in winter, can be stored at low temperature for a long time without feeding, and are convenient to obtain, solving the problem that Exorista sorbillans Wiedemann cannot be normally propagated in winter and overcoming the problem of difficulty in obtaining fly eggs under the condition of no host. By attracting a large number of flies to lay eggs, the rapid in vitro cultivation of Exorista sorbillans Wiedemann is successfully realized, and adult flies are obtained. Among them, the addition of 10% hemolymph of the sixth day of the fifth instar of Bombyx mori can reach a maximum adult fly rate of 69.6%.

[0038] (2) The method of the present invention can rear Exorista sorbillans Wiedemann throughout the year. Different living species are selected at different times, and it is designed to attract flies to lay eggs at 28 °C, solving the problem that a large number of eggs cannot be laid in non-suitable seasons and without natural hosts.

[0039] (3) The cultivation method of the present invention is time-saving and simple to operate. After placing the fly eggs, no other operations are required, and just wait for the fly maggots to pupate. The link of regularly feeding the host in the conventional rearing of tachinid flies is removed, and the hemolymph of the sixth day of the fifth instar of Bombyx mori, which is the most suitable for adding to the culture medium, is screened out. This culture medium significantly reduces the development time of fly maggots. The parasitic effect of the tachinid flies obtained by the present invention has no significant difference from natural parasitism. Description of the Drawings

[0040] Figure 1 It is a drawing of Exorista sorbillans Wiedemann laying eggs. Among them, A is a drawing of the fifth instar larvae of Bombyx mori attracting Exorista sorbillans Wiedemann to lay eggs, B is a drawing of the pupae of Bombyx mori attracting Exorista sorbillans Wiedemann to lay eggs, C is a drawing of the larvae of Tenebrio molitor attracting Exorista sorbillans Wiedemann to lay eggs, D is a drawing of the larvae of Exorista sorbillans Wiedemann attracting Exorista sorbillans Wiedemann to lay eggs, and E is a drawing of the pupae of Antheraea pernyi attracting Exorista sorbillans Wiedemann to lay eggs;

[0041] Figure 2 It is a growth drawing of the eggs of Exorista sorbillans Wiedemann in the BM + 10% hemolymph of the sixth day of the fifth instar of Bombyx mori medium in Example 2. Among them, A is a drawing of the eggs of Exorista sorbillans Wiedemann growing to the third instar larvae in the medium, and B is a drawing of the eggs of Exorista sorbillans Wiedemann growing to pupation;

[0042] Figure 3 It is partial fly pupae and female flies obtained from the medium added with 10% hemolymph of the sixth day of the fifth instar of Bombyx mori (i.e., the tachinid flies obtained by in vitro culture with the optimal medium). Detailed Embodiments

[0043] The present invention will be further explained and illustrated below in conjunction with specific examples.

[0044] For a better understanding of the operation process of the invention, the methods for obtaining fly eggs and preparing the culture medium will be described in detail below. The cultivation and propagation effects of the invention on Exorista sorbillans Wiedemann are proved by the fly egg hatching experiment under different species and the experiments on the development time, pupal weight, emergence rate and parasitism rate of fly maggots under the hemolymph of Bombyx mori at different times.

[0045] Original fly species: The original fly pupae were collected in the wild by the Key Laboratory of Sericultural Genetic Improvement, Ministry of Agriculture and Rural Affairs, College of Biotechnology, Jiangsu University of Science and Technology, and the adult flies were propagated indoors. The fly cage contained 100 - 200 adult flies (length × width × height, 50 cm × 40 cm × 60 cm).

[0046] Egg-laying carriers: Plastic film, fifth-instar silkworm larvae and pupae (Jingsong × Haoyue, fifth-instar silkworm larvae are restricted, and the growth stage of pupae is not restricted), Tenebrio molitor larvae (any commercially available, the size of larvae is not restricted), Antheraea pernyi pupae (any commercially available), mature larvae of Exorista sorbillans (the larvae of Exorista sorbillans are restricted to the maggots that have crawled out after maturity, and the size of larvae is not restricted).

[0047] Selection of species to attract Exorista sorbillans to lay eggs: During the silkworm rearing period, fifth-instar silkworms or silkworm pupae can be selected. During the non-silkworm rearing period, Tenebrio molitor larvae and Antheraea pernyi pupae are purchased from the market, or mature larvae of Exorista sorbillans. Among them, the optimal one is Antheraea pernyi pupae.

[0048] Culture medium supplies: Skim milk (purchased from Bright Dairy products), silkworm hemolymph (preferably the hybrid large silkworms - Jingsong × Haoyue, and the optimal is the sixth day of the fifth instar), Gentamicin sulfate injection (concentration 4 mg / ml), absorbent cotton (purchased from commercially available medical absorbent cotton).

[0049] Data analysis: One-way ANOVA was used to analyze the data by GraphPad Prism 8.0. Among them, hatching rate (%) = number of first-instar larvae / total number of eggs; pupation rate (%) = number of fly pupae / total number of eggs; adult fly emergence rate (%) = number of adult flies / total number of eggs; maggot development time (D) = maggot pupation time - egg collection time; parasitism rate (%) = number of parasitized spots / total number of eggs.

[0050] Example 1

[0051] Collecting eggs of Exorista sorbillans and investigating the subsequent hatching rate of fly eggs

[0052] The specific operation of this method is as follows:

[0053] Step 1: Seven days after the emergence of adult flies propagated by laboratory parasitism (the flies start to lay eggs on the third day after emergence, and about seven days after emergence is the peak egg-laying period). At 1 pm, with the indoor temperature of 28°C, a plastic film (with an area of about 1 / 4 of the bottom area of the fly cage), two silkworms, two silkworm pupae, two Antheraea pernyi pupae, seven Tenebrio molitor larvae and four mature larvae of Exorista sorbillans were respectively placed in the fly cage.

[0054] Step 2: After 20 minutes, the silkworms were taken out, and the other ones were taken out after extending the parasitism time to 2 hours, and the density of fly eggs on the surface of the egg-laying carrier was observed.

[0055] Step 3: At 9:00 am the next day, collect the fly eggs described in Step 2. For the surface-parasitized eggs of species other than the larvae of Exorista sorbillans Wiedemann, use forceps to remove and collect them in a 1.5 ml EP tube. Since the larvae of Exorista sorbillans Wiedemann have pupated and are small in size, soak them in clear water for 10 s, gently rub them while wearing gloves, and remove the fly eggs on the surface of the fly pupae and collect them in a 1.5 ml EP tube.

[0056] Step 4: Preparation of basic medium (BM): Add 30 ml of skim milk and 0.9 g of sucrose to a beaker and stir to dissolve. In another beaker, add 2.7 g of yeast extract and 9.9 ml of sterilized water and stir to dissolve. Divide absorbent cotton into 15 mg per piece, knead it into a ball and place it in a small aluminum box, cover and seal. Autoclave the above two beaker solutions and absorbent cotton balls at 115 °C for 10 min. After cooling, mix the milk-sucrose and yeast extract solutions in a laminar flow hood. Wipe the surface of the egg with 75% alcohol and let it dry. Crack the egg into a sterile petri dish, use a disposable syringe to aspirate 5.4 ml of egg yolk and add it to the just-prepared mixed solution. Then add 97.8 μl of gentamicin sulfate solution and stir evenly. Aliquot into 24-well plates, with 400 μl of liquid medium + 15 mg of absorbent cotton in each well.

[0057] Step 5: Disinfect the fly eggs collected in Step 3 with 70% ethanol for 5 min, rinse twice with sterile water, use forceps to pick up the fly eggs and put them into the medium described in Step 4, cover the lid, incubate in an incubator at 26 °C, 75% RH, 0L:24D. After 4 days, observe and count the hatching rate of fly maggots under a microscope (where the fly eggs hatch into first-instar fly maggots, and the hatching rate of fly eggs is judged according to the number of first-instar fly maggots). The results are shown in Table 1.

[0058] Table 1: Hatching rate of fly maggot eggs after taking eggs from the surfaces of different species

[0059]

[0060] Note: No same letters in the same column represent significant differences. a, b, c represent significant differences (P < 0.05).

[0061] The number of fly eggs obtained in the plastic film was 2, which did not meet the quantity requirement for the next collection and passage. There were excessive fly eggs on the body surfaces of the other five insects, such as Figure 1As shown, the mealworms are highly active with a hard and smooth body surface. The fly eggs parasitized on their surface are prone to fall off, and the number of collected fly eggs is lower than that of other insects. Fly eggs were collected from the surfaces of five types of insects and incubated. As can be seen from Table 1, there is no significant difference in the hatching rate of fly eggs taken from silkworm pupae, tussah pupae, and mature larvae of Exorista sorbillans parasitized on silkworm pupae compared with that of the fifth-instar silkworms; the hatching rate of fly eggs taken from the surface of mealworm larvae is relatively low, probably because the mealworm larvae are highly mobile and have a hard epidermis, which will cause stronger mechanical damage to the fly eggs during egg collection; among them, the tussah pupae are in diapause in winter, can be stored at low temperature for a long time without feeding, and are convenient to use. Considering the above aspects, when there is no natural host (even in cold weather), the tussah pupa is the most suitable species to attract Exorista sorbillans to lay eggs.

[0062] Example 2

[0063] Using tussah pupae to investigate the number of eggs laid by tachinid flies at different temperatures and their hatching rate

[0064] The specific operation steps are as follows:

[0065] Take the flies that emerged simultaneously from being parasitized in the silkworm body (8 female flies and 8 male flies) and raise them in a fly cage (26±1°C), with 3 parallel groups. When it is 7 days after emergence, adjust the temperature to 24°C, 28°C, and 32°C respectively. After the temperature is constant, treat for 1 h, then place 2 tussah pupae in each fly cage respectively. After parasitism for 2 h, take them out and count the number of fly eggs in each group. Conduct three repeated experiments. Treat the fly eggs collected under the conditions of 28°C and 32°C and then put them into the culture medium for cultivation. Other operations are the same as in Example 1, and count the hatching rate of the fly eggs.

[0066] Table 2: The egg-laying situation and hatching rate of Exorista sorbillans on the surface of tussah pupae at different temperatures

[0067]

[0068] Note: Different letters in the same column indicate significant differences, and A, B, and C indicate extremely significant differences (P<0.001).

[0069] As can be seen from Table 2, when the temperature is 24°C, the adult flies lay fewer eggs. When the temperature is increased to 28°C, the number of eggs laid by the adult flies is significantly higher than that at 24°C. When the temperature is increased to 32°C, the number of fly eggs laid by the adult flies is significantly higher than that at 28°C. However, the hatching rate of the fly eggs at 32°C is significantly lower than that at 28°C. Considering comprehensively, 28°C is the appropriate temperature for collecting fly eggs.

[0070] Example 3

[0071] Using the culture medium described in the present invention to conduct in vitro culture of the collected fly eggs

[0072] The specific operation steps of this method are as follows:

[0073] Step 1, Collection and treatment of fly eggs: Collect fly eggs using tussah silkworm pupae as described in Example 1.

[0074] Step 2, Preparation of culture medium:

[0075] First, treat the hemolymph of silkworms: 1) Rinse the silkworms of Jing Song × Hao Yue at the second day of the fifth instar (L5D2), the fourth day of the fifth instar (L5D4), the sixth day of the fifth instar (L5D6), 2 weeks after mounting (2D), and 4 weeks after mounting (4D) with pure water respectively, and heat in a water bath at 60 °C for 10 min; 2) Under sterile conditions, wipe the body surface of the above silkworms with an alcohol cotton ball, cut open the first pair of abdominal feet of the silkworms with a clean scissors, collect the hemolymph of the silkworms into sterile EP tubes respectively, store at -20 °C, and take out and melt when in use.

[0076] Preparation of BM medium: As described in Example 1.

[0077] Except for the control BM group, add 5.5 ml of hemolymph of silkworms at the second day of the fifth instar (L5D2), the fourth day of the fifth instar (L5D4), the sixth day of the fifth instar (L5D6), 2 weeks after mounting (2D), and 4 weeks after mounting (4D) respectively to the others, stir and mix evenly. Add 97.8 μl of gentamicin sulfate to the culture medium of the control BM group, and add 108.8 μl of gentamicin sulfate injection (working concentration 8 μg / ml) to the culture medium of each of the other groups, and mix evenly to obtain a liquid culture medium. Put the sterilized absorbent cotton into a 24-well plate, one for each well, add 400 μl of the liquid culture medium to each well, and spread evenly. Each group has three replicates.

[0078] Step 3, Incubation and culture of fly eggs: Put the fly eggs described in Step 1 into the culture medium described in Step 2 with tweezers, one fly egg per well, cover the lid and seal it with a sealing film, then place it at 70% RH in an incubator at 26 °C for 0L:24D culture, and count the pupation situation of the fly eggs and the growth time of the maggots (the time from the fly eggs developing to pupation). The results are shown in Table 3.

[0079] Step 4, Collection and treatment of fly pupae: Collect the fly pupae obtained in Step 3 into a clean petri dish uniformly, place it at 75% RH in the dark at 26 °C (0L:24D), and obtain the emerged flies after 12 - 13 days. Count the emergence rate of the flies. The results are shown in Table 3.

[0080] Step 5, Rearing of emerged flies: Put the artificially cultured Exorista sorbillans emerged flies obtained in Step 4 into an indoor fly cage (length × width × height, 35 cm × 35 cm × 35 cm), rear at 26 °C with 12L:12D, and feed 10% honey water every day.

[0081] Table 3: Effects of hemolymph of silkworms at different stages on the growth and development of maggots of Exorista sorbillans

[0082]

[0083]

[0084] Note: Different letters in the same column indicate significant differences. a, b, and c indicate significant differences (P < 0.05).

[0085] As can be seen from Table 3, the average pupation rate was relatively high in the groups with Bombyx mori hemolymph added with L5D4 and L5D6. And there was a significant difference in the average pupation rate between the BM + L5D6 group and the BM group. Both L5D6 and the hemolymph of Bombyx mori two days after mounting could minimize the development duration of Exorista sorbillans larvae, being closer to the parasitic development time of Exorista sorbillans on the silkworm body (9 - 12 days). In addition, the fly maggot to adult fly rate of the BM + L5D4 group and the BM + L5D6 group was significantly better than that of the BM group, and the hemolymph of Bombyx mori at other stages did not significantly increase the adult fly rate. Based on the above results, adding L5D6 hemolymph of Bombyx mori to BM could minimize the development duration of the tachinid fly while ensuring a high adult fly rate. Therefore, the optimal choice was the hemolymph of L5D6 Bombyx mori. The culture results of BM + L5D6 are as Figure 2 、 Figure 3 shown. The basic epidermis of the fly pupae cultured in vitro was round and the morphology was normal. The appearance of the female flies after eclosion was good. At the same time, compared with the existing in vitro culture effect of Exorista sorbillans on Bombyx mori, the specific culture medium and culture method used in the present invention shortened the development time of fly maggots. The development time of fly maggots was similar to the natural parasitic conditions and had a very high adult fly rate.

[0086] Example 4

[0087] Effect of Different Contents of L5D6 Hemolymph of Bombyx mori in the Culture Medium on the Pupation Rate of Tachinid Flies

[0088] The operation steps of this method are as follows:

[0089] Step 1: Obtain fly eggs by the method of Example 1. Adjust the temperature of the incubator to 28 °C, keep other processes unchanged, and place Antheraea pernyi pupae in the fly cage for excessive parasitism.

[0090] Step 2: Prepare culture media BM and BM + L5D6 by the method of Example 3, where the addition amounts of Bombyx mori hemolymph are 5%, 10%, and 20% of the total volume of the liquid culture medium respectively.

[0091] Step 3: Collect, process, and culture fly eggs in the same way as in Example 3.

[0092] Step 4: Collect, process, and culture fly pupae in the same way as in Example 3. Statistically analyze the fly pupa rates under BM and three concentrations of Bombyx mori hemolymph (L5D6). The results are shown in Table 4.

[0093] Table 4: Pupation Rates of Exorista sorbillans on Bombyx mori under Different Concentrations of L5D6 Hemolymph of Bombyx mori

[0094]

[0095]

[0096] Note: The same letters in the same column indicate significant differences, a and b indicate significant differences (P<0.05).

[0097] As can be seen from Table 4, there is no significant difference in the average pupation rate between the group with 5% L5D6 silkworm hemolymph and the BM group. When the concentration of silkworm hemolymph increases to 10% of the total volume of the culture medium, the pupation rate increases significantly. When the concentration of silkworm hemolymph increases to 20% of the total volume of the culture medium, the pupation rate is no significant difference from the BM+10% L5D6 group. Considering the cost factor, the optimal proportion of L5D6 silkworm blood addition is 10% of the total liquid culture medium.

[0098] Example 5

[0099] Comparison of the weight of pupae and parasitic conditions of adult flies of the silkworm parasitic flies cultured in vitro and naturally parasitized

[0100] The specific steps of this method are as follows:

[0101] Step 1: Comparison of fly pupa weights. Collect 38 fly pupae obtained from BM in Example 3, 48 fly pupae obtained from BM+L5D6, and 40 fly pupae randomly selected after natural parasitism of silkworms in the silkworm room (2-3 eggs per silkworm), weigh and record them one by one, calculate the average weight, and place them in a completely dark 26°C constant temperature incubator. The results are shown in Table 5. Among them, natural parasitism conditions refer to: after the silkworm chasing fly lays eggs on the surface of the silkworm, the silkworm is raised normally at about 26°C until the fly maggots crawl out when the silkworm is on the cluster.

[0102] Step 2: Comparison of parasitism rate. Prepare three fly cages (length × width × height, 35cm × 35cm × 35cm), and put the three groups of adult flies obtained from the emergence of fly pupae in step 1 respectively. After 7 days of emergence of the first parasitic fly in each group, make the parasitism time point and temperature conditions during parasitism the same as those in Example 1. Put 3 silkworms in each group of fly cages and take them out after parasitism for 20 minutes, ensuring that there are 10-20 fly eggs on each silkworm. The silkworms are grouped and marked and fed normally (26±1℃). After four days of parasitism, the total number of eggs and parasitic spots are counted. The results are shown in Table 5.

[0103] Table 5: Pupa weight and adult fly parasitism rate of in vitro cultured and naturally parasitic flies

[0104]

[0105] Note: The same letters in the same column indicate significant differences, where A, B, and C indicate extremely significant differences (P<0.0001), and a and b indicate significant differences (P<0.05).

[0106] As can be seen from Table 5, the pupal weight of fly pupae in the natural parasitism group > BM + L5D6 group > BM group. The culture of tachinids with the addition of L5D6 silkworm blood is superior to the BM group; the parasitism rate of the artificially cultured BM group is significantly lower than that of the natural parasitism group, but the parasitism rate of the BM + L5D6 group has no obvious difference from that of the natural parasitism group, and the reproductive performance of adult flies under in vitro culture is close to that under natural parasitism.

[0107] Based on the results of the above examples, the present invention overcomes the in vitro culture and subculture work of Exorista sorbillans under winter or host-free conditions, shortens the in vitro cultivation time of tachinids, and maintains the body size and parasitism rate of Exorista sorbillans to a certain extent. The species, oviposition temperature, and the addition of silkworm hemolymph at a fixed stage and its proportion in the culture medium involved in the above examples for attracting Exorista sorbillans to lay eggs are all within the scope of protection of the present invention.

Claims

1. A method for culturing Exorista sorbillans in vitro, characterized in that, It includes the following steps: Step 1, collection and treatment of fly eggs: Parasitic passaged adult flies emerge for 7 days, indoor temperature is 28 °C. Put plastic film and tussah pupae into the fly cage respectively; take them out after 2 h and observe the density of fly eggs on the surface of tussah pupae; the next morning, collect fly eggs and remove the parasitic eggs on the surface of tussah pupae. Step 2, preparation of culture medium: First, treat silkworm hemolymph: 1) Rinse the fifth instar sixth-day Jing Song × Hao Yue silkworms with pure water and heat them in a water bath at 60 °C for 10 min; 2) Under sterile conditions, wipe the surface of the above silkworms with an alcohol cotton ball, cut open the first pair of abdominal feet of the silkworms, and collect the silkworm hemolymph. Stir 30 ml of skim milk and 0.9 g of sucrose to dissolve into a milk-sucrose solution; stir 2.7 g of yeast extract and 9.9 ml of sterilized water to dissolve into a yeast extract solution; divide the absorbent cotton into 15 mg / each and knead into balls; put the above solutions and absorbent cotton into a high-pressure sterilizer at 115 °C for 10 min. After cooling, mix the milk-sucrose solution and the yeast extract solution in a laminar flow hood. Wipe the surface of the egg with 75% alcohol and dry it. Absorb 5.4 mL of egg yolk and add it to the just-prepared mixed solution. Add 5.5 mL of the fifth instar sixth-day silkworm hemolymph, and then add 108.8 μL of gentamicin sulfate injection with a working concentration of 8 μg / mL and stir evenly. Aliquot it into 24-well plates, with 400 μL of liquid culture medium + 15 mg of absorbent cotton in each well. Step 3, hatching and culturing of fly eggs: Put the fly eggs described in Step 1 into the culture medium described in Step 2 with tweezers, 1 fly egg per well. After covering the lid, seal it with sealing film and place it in an incubator at 70% RH and 26 °C for 0L:24D culture. Count the pupation situation of fly eggs and the growth time of maggots. Step 4, collection and treatment of fly pupae: Uniformly collect the fly pupae obtained in Step 3 in a clean petri dish and place them in a completely dark place at 75% RH and 26 °C. Adult flies will emerge after 12 - 13 days. Count the emergence rate of adult flies. Step 5, rearing of adult flies: Put the artificially cultured Exorista sorbillans adult flies obtained in Step 4 into an indoor fly cage with dimensions of length × width × height of 35 cm × 35 cm × 35 cm, and rear them at 26 °C with a photoperiod of 12 L:12 D. Feed 10% honey water every day.

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