A method for cultivating insect symbiotic bacteria

Abstract

The present invention belongs to the field of microbial culture technology and discloses a method for culturing insect symbiotic bacteria. The method comprises the following steps: Step S1, preparing an insect cell feeder layer; Step S2, collecting insect eggs; Step S3, disinfecting the insect eggs; Step S4, crushing the insect eggs and inoculating and culturing the symbiotic bacteria; and Step S5, isolating and expanding the insect symbiotic bacteria. The present invention utilizes an insect cell feeder layer to cultivate insect symbiotic bacteria, and utilizes cytokines or other unknown components secreted by the feeder layer cells to promote the growth and reproduction of symbiotic bacteria that are difficult to culture directly.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial cultivation, and particularly relates to a method for cultivating insect symbiotic bacteria. Background Art

[0002] Insect symbionts live in mutually beneficial relationships with insects. Many of these bacteria, which maintain close symbiotic relationships with their host insects, are difficult to culture directly using microbial culture media. These bacteria rely on specific nutrients or growth signals within their hosts, and conventional culture media cannot meet their demanding growth requirements, leading to in vitro culture failure. This limits in-depth research into the functions and potential applications of these symbionts.

[0003] By preparing an insect cell feeder layer and utilizing the cytokines and other components released by the insect cell feeder layer, it is possible to simulate the microenvironment within the insect body, thereby promoting the growth and reproduction of insect symbiotic bacteria. This method may be used to cultivate some difficult-to-cultivate insect symbiotic bacteria. Summary of the Invention

[0004] The object of the present invention is to provide a method for culturing insect symbiotic bacteria using an insect cell feeder layer, so as to solve the problem in the prior art that some insect symbiotic bacteria are difficult to culture on conventional culture media.

[0005] To achieve the above object, the present invention provides a method for culturing insect symbiotic bacteria, comprising the following steps:

[0006] Step S1: Preparation of insect cell feeder layer.

[0007] Insect cells (e.g., Sf9 cells) are plated onto a culture dish and cultured in a 27°C incubator until the cells reach 70% confluence. Then, they are treated with mitomycin C at a final concentration of 100 μg / mL for 2 hours. After treatment, the culture medium is replaced with fresh insect cell culture medium. This treatment results in a monolayer of metabolically active but non-dividing insect cells, forming a feeder layer.

[0008] Step S2: collecting insect eggs.

[0009] After mating, the female adults are placed on a suitable host plant (such as rice) to lay eggs. After 2-5 days, the plant tissue is dissected and the eggs are collected.

[0010] Step S3: disinfection of insect eggs.

[0011] Disinfect the insect eggs collected in step S2 with 0.25% sodium hypochlorite solution for 5 minutes, then with 75% alcohol for 5 minutes. Finally, rinse three times with 1x PBS (phosphate-buffered saline) to remove residual disinfectant.

[0012] Step S4: crushing the insect eggs and inoculating and culturing the symbiotic bacteria.

[0013] Under sterile conditions, mince the eggs disinfected in step S3 with sterile ophthalmic scissors or crush them with a sterile grinding rod. Add an appropriate amount of cell culture medium (e.g., fresh insect cell culture medium) and dissociate the symbiotic bacteria from the eggs by pipetting or shaking. Then, use a pipette to transfer the culture suspension containing the free symbiotic bacteria to the cell culture dish containing the insect cell feeder layer prepared in step S1 for co-cultivation.

[0014] Step S5: Isolation and expansion of insect symbiotic bacteria.

[0015] After the symbiotic bacteria in the co-culture system in step S4 have grown, aspirate or pick the growth and streak it onto a suitable bacterial or fungal culture plate (e.g., a YPD plate). After incubating the plate at a suitable temperature, single colonies with good morphology are picked and inoculated into the appropriate liquid culture medium for shake culture to obtain a pure culture.

[0016] The method for cultivating insect symbiotic bacteria of the present invention has the following significant advantages over the prior art:

[0017] This invention uses insect cell feeder layers to cultivate insect symbiotic bacteria, leveraging cytokines or other unknown components secreted by the feeder cells to promote their growth. By simulating the internal environment of insects, this method provides an effective in vitro culture pathway for host cell-dependent symbiotic bacteria that are difficult to grow in conventional culture media, facilitating subsequent research and utilization of these symbiotic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive work, among which:

[0019] Figure 1 This is a comparison diagram of the culture states of the symbiotic bacteria under culture conditions with and without an Sf9 cell feeder layer in an embodiment of a culture method for insect symbiotic bacteria of the present invention;

[0020] Note: In the figure, A and B are the initial state and 48 hours later of symbiotic fungi cultured on Sf9 cell feeder layer, respectively; in the figure, C and D are the initial state and 48 hours later of symbiotic fungi cultured without Sf9 cell feeder layer, respectively; the circled ones are symbiotic fungi; the scale bar in the figure is 100 μm.

[0021] Figure 2 This is a photograph of the symbiotic fungal colonies isolated and purified on a YPD plate in an embodiment of a method for culturing insect symbiotic bacteria of the present invention. DETAILED DESCRIPTION

[0022] The following describes in detail the implementation details of the method for cultivating insect symbiotic bacteria of the present invention in conjunction with specific embodiments, so that ordinary technicians in the relevant technical field can implement the present invention.

[0023] Example 1: Cultivation of symbiotic fungi of brown planthopper

[0024] Step S1: Preparation of insect cell feeder layer using Sf9 cells

[0025] Sf9 cells were treated with Sf-900 TM II SFM cell culture medium was subcultured in a 27°C cell culture incubator. When the feeder layer needs to be prepared, the cells were seeded into a cell culture dish, the seeding density was adjusted, and the cells were cultured in a 27°C incubator until the cell confluence reached 70%. Then, mitomycin C was added to the culture medium to a final concentration of 100 μg / mL and treated for 2 hours. After the treatment, the culture medium containing mitomycin C was discarded, the cell layer was rinsed 3 times with 1× PBS, and finally replaced with fresh Sf-900. TM II SFM cell culture medium.

[0026] Step S2: Collection of brown planthopper eggs

[0027] The paired brown planthopper ( Nilaparvata lugens ) Female adults are placed on rice seedlings to lay eggs. Two to five days after egg laying, rice leaf sheaths are dissected and the brown planthopper eggs collected.

[0028] Step S3: Disinfection of brown planthopper eggs

[0029] The collected brown planthopper eggs were first disinfected with 0.25% sodium hypochlorite solution for 5 minutes, then with 75% alcohol for 5 minutes, and finally rinsed three times with 1× PBS.

[0030] Step S4: Crushing of brown planthopper eggs and inoculation of symbiotic fungi

[0031] Cut the sterilized eggs into pieces with sterile ophthalmic scissors and add appropriate amount of fresh Sf-900 TM II SFM cell culture medium to release the symbiotic fungi from the eggs. Use a pipette to inoculate the suspension containing the symbiotic fungi into the cell culture dish containing the Sf9 cell feeder layer prepared in step S1. Optionally, add penicillin at a final concentration of 100 U / mL and streptomycin at a final concentration of 100 μg / mL to inhibit bacterial contamination. Incubate in a 27°C incubator. A control group is inoculated into a culture dish without a feeder layer. Observe after 48 hours of culture (see [see "Cultivate the fungi suspension in a 27°C incubator"]). Figure 1 ), it can be seen that the fungi in the feeder layer group grew well, while the fungi in the group without feeder layer grew inhibited.

[0032] Step S5: Isolation and expansion of insect symbiotic fungi

[0033] The symbiotic fungi samples grown in the feeder layer culture system in step S4 were streaked onto YPD solid medium plates. The plates were placed in a 27°C incubator for 3-5 days until single colonies were grown (see Figure 2 ). A single colony was picked and inoculated into YPD liquid medium and cultured in a shaking incubator at 27°C. The obtained pure culture was subjected to ITS sequence PCR and sequencing identification. The results showed that the symbiotic fungus was Moesziomyces (Ustilago mori).

[0034] This invention uses insect cell feeder layers to cultivate insect symbiotic bacteria, leveraging cytokines or other unknown components secreted by the feeder cells to promote their growth. By simulating the internal environment of insects, this method provides an effective in vitro culture pathway for host cell-dependent symbiotic bacteria that are difficult to grow in conventional culture media, facilitating subsequent research and utilization of these symbiotic bacteria.

[0035] Although specific embodiments of the present invention have been described above, those skilled in the art will appreciate that these specific embodiments are merely illustrative, and that those skilled in the art may omit, substitute, and modify the details of the methods and systems described above without departing from the principles and spirit of the present invention. For example, combining the above method steps to perform substantially the same functions in substantially the same manner to achieve substantially the same results falls within the scope of the present invention. Accordingly, the scope of the present invention is limited solely by the appended claims.

Claims

1. A method for cultivating insect symbiotic bacteria, characterized in that: The following steps are involved: Step S1, Preparation of an Insect Cell Feeder Layer: Sf9 cells were seeded into a cell culture dish and cultured in a 27°C incubator until the cell confluence reached 70%. The cells were then treated with mitomycin C at a final concentration of 100 µg / mL for 2 hours and then replaced with fresh insect cell culture medium to obtain the Sf9 cell feeder layer. Step S2, collection of insect eggs: the paired brown planthopper ( Nilaparvata lugens ) The female adult insects are inoculated on their host plant rice to lay eggs, and after 2 to 5 days, the plant tissues are dissected to collect the brown planthopper eggs; Step S3, disinfection of insect eggs: the insect eggs collected in step S2 were first disinfected with 0.25% sodium hypochlorite solution for 5 minutes, then disinfected with 75% alcohol for 5 minutes, and finally rinsed three times with 1× PBS; Step S4, crushing insect eggs and inoculating symbiotic bacteria: the eggs disinfected in step S3 are chopped up with ophthalmic scissors or crushed with a small grinding rod, an appropriate amount of cell culture medium is added to release the symbiotic bacteria in the eggs, and the released symbiotic bacteria are inoculated into a cell culture dish containing an Sf9 cell feeder layer prepared in step S1 using a pipette for culture; Step S5, Isolation and expansion of insect symbiotic bacteria: The symbiotic bacteria grown in step S4 are streaked on a YPD medium plate, and then a single colony is selected for shake culture; Wherein, the symbiotic bacteria belongs to the genus Ustilago officinalis ( Moesziomyces of fungi.

2. The method for cultivating insect symbiotic bacteria according to claim 1, characterized in that: The cell culture medium in step S1 and step S4 is Sf-900 TM IISFM cell culture medium.

3. The method for cultivating insect symbiotic bacteria according to claim 1, characterized in that: The crushing method in step S4 is to use ophthalmic scissors to cut into pieces or to use a small grinding rod to crush into pieces.

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