Bionic culture method and system for ghost moth larvae

Abstract

The invention provides a bionic culture method and system for hepialus armoricanus larvae, and belongs to the technical field of hepialus armoricanus larva culture. The method overcomes the problems of insufficient microbial diversity and poor environmental adaptability during artificial feeding of hepialus armoricanus larvae in the prior art by constructing a mixed culture medium and simulating a wild environment, and comprises the following steps: treating a biological sample of intestinal tracts of wild hepialus armoricanus larvae, extracting a target strain and preparing a fungal spore suspension; the method comprises the following steps: mixing alpine meadow herbaceous plants, alpine shrub dwarf plants and alpine meadow moss plants, sterilizing part of materials, and inoculating fungal spore suspension to form a mixed culture medium; the environment parameters of the natural habitat are monitored in real time, and the culture parameters in the culture cabin are adjusted in real time according to the collected data, so that the high consistency of the culture environment and the natural habitat is ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of bat moth larvae cultivation, and in particular relates to a bat moth larvae bionic cultivation method and system. Background Art

[0002] As an important insect resource, bat moth larvae are widely distributed in the Qinghai-Tibet Plateau and surrounding areas of China. In their natural habitat, these larvae rely on specific alpine meadow plants and complex microbial communities for growth and development. Traditionally, bat moth larvae have attracted much attention due to their symbiotic relationship with Cordyceps sinensis, which has important value in the fields of medicine and scientific research. However, with the increasing interest in this unique organism, how to successfully cultivate bat moth larvae under artificial conditions has become an urgent problem to be solved.

[0003] At present, the technology of artificially rearing bat moth larvae is still in its infancy and there are many challenges. Existing methods generally fail to fully simulate the complex ecological conditions in the natural environment, especially in terms of microbial ecology. Simple artificial rearing methods fail to provide sufficient microbial diversity and a complex ecological environment, resulting in the inability of certain specific bacterial communities in the larval intestine to colonize normally, which in turn affects their health and growth and development. In addition, although the strict disinfection measures commonly used in artificial rearing environments reduce the presence of pathogens, they also eliminate many microorganisms that are beneficial to the larvae, further weakening the larvae's immunity and adaptability.

[0004] Therefore, it is particularly important to develop a biomimetic culture method that can highly restore the wild environment, maintain a healthy microbial community, and promote the healthy growth of larvae. Summary of the invention

[0005] The invention overcomes the shortcomings of the prior art and provides a bionic culture method and system for bat moth larvae.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a bionic culture method for bat moth larvae, comprising the following steps:

[0007] S1. Process biological samples from the intestines of wild bat moth larvae, extract target strains, and prepare fungal spore suspensions;

[0008] S2, washing, cutting and mixing leaves of alpine meadow herbaceous plants, young branches and leaves of alpine shrub dwarf plants and stems and leaves of alpine meadow mosses to obtain mixed plant materials;

[0009] S3, inoculating the fungal spore suspension onto the mixed plant material to form a mixed culture matrix;

[0010] S4, simulated wild environment;

[0011] S5, culturing the bat moth larvae in a mixed culture matrix;

[0012] Among them, the target strains include one or more of Penicillium, Aspergillus, Gibberellic acid and Trichoderma.

[0013] Furthermore, the processing of the biological sample from the intestine of wild bat moth larvae in step S1 includes:

[0014] S11, preparing potato dextrose agar medium;

[0015] S12, dissecting wild bat moth larvae, taking their intestinal contents, and inoculating them on potato dextrose agar medium;

[0016] S13, placing the inoculated culture medium in a constant temperature incubator, setting the temperature to 19-21°C, the humidity to 85%-90%, and culturing for 5-7 days;

[0017] S14. Pick a single colony and transfer it to a new potato dextrose agar medium for purification three or more times to obtain the target strain.

[0018] Furthermore, the method for preparing the fungal spore suspension in step S1 is: removing mature fungal spores from the target strain, adding sterile physiological saline, shaking evenly, and adjusting the spore concentration to 1×10 6 -10 8 / mL to obtain a fungal spore suspension.

[0019] Furthermore, the mixed plant material in step S2 contains 30-50% alpine meadow herbaceous plants, 20-40% alpine shrub dwarf plants and 20-40% alpine meadow mosses; wherein the alpine meadow herbaceous plants include one or more of Oxytropis purpurogena, Polygonum villosa and Pedullis dwarfis; the alpine shrub dwarf plants include one or more of Rhododendron microphylla, Potentilla fruticosa and Ceratoides cuspidatus; the alpine meadow mosses include one or more of Pseudocematophyte, Psoralea corylifolia and Black moss.

[0020] Furthermore, step S2 also includes: sterilizing 20-60% of the mixed plant material; in step S3, inoculating the fungal spore suspension into the sterilized mixed plant material, and mixing the inoculated mixed plant material with the remaining unsterilized plant material to obtain a mixed culture matrix.

[0021] Furthermore, the method of simulating a wild environment in step S4 includes:

[0022] S41. Real-time monitoring of environmental parameters in the natural habitat of bat moth larvae;

[0023] S42, adjusting the culture parameters in real time according to the environmental parameters so that the culture parameters are synchronized with the environmental parameters;

[0024] Both environmental parameters and culture parameters include temperature, humidity, light, air pressure, wind speed, rain, pH, carbon dioxide concentration, oxygen concentration and ultraviolet radiation.

[0025] Further, step S5 includes:

[0026] S51, culturing the bat moth larvae in a mixed culture matrix at a larval density of 200-300 per square meter;

[0027] S52. During the culture process, the culture medium is replaced regularly, with a frequency of once every 3-5 days.

[0028] Furthermore, the bionic culture method for bat moth larvae provided by the present invention further comprises step S6:

[0029] monitoring the microbial composition of the larval gut;

[0030] The growth rate and mortality of the larvae were monitored, and the growth, development and health status of the larvae were recorded.

[0031] Another technical solution provided by the present invention is a bionic culture system for bat moth larvae, based on the above method, comprising:

[0032] Environmental parameter collection module, used to collect environmental parameters of the natural habitat of bat moth larvae;

[0033] A culture chamber for cultivating bat moth larvae;

[0034] The environmental parameter transmission module is connected to the environmental parameter acquisition module by signal, and is used to transmit the collected environmental parameters to the culture chamber control module;

[0035] A culture chamber environmental parameter monitoring module is used to monitor the culture parameters in the culture chamber;

[0036] The culture chamber control module is used to receive the environmental parameters transmitted by the environmental parameter transmission module and adjust the culture parameters of the culture chamber according to the collected environmental parameters.

[0037] Further, the environmental parameter transmission module includes a signal tower A and a signal tower B connected by signals;

[0038] Further, the environmental parameter acquisition module includes a temperature sensor, a humidity sensor, a light sensor, an air pressure sensor, a wind speed sensor, a pH sensor, a carbon dioxide sensor, an oxygen sensor and an ultraviolet sensor which are integrated and installed in the signal tower A;

[0039] Furthermore, the culture chamber control module includes an automatic control system based on a programmable logic controller, and actuators connected to the control system including a thermostat, a humidifier, a dehumidifier, an LED lamp, a gas flow controller, a small fan and an ultraviolet lamp.

[0040] The present invention solves the defects existing in the background technology and has the following beneficial effects:

[0041] (1) The present invention solves the problems of insufficient microbial diversity and poor environmental adaptability in the prior art of artificially rearing bat moth larvae by constructing a mixed culture matrix and simulating a wild environment to be as close to wild conditions as possible. Cordyceps sinensis containing fungi is considered to be closer to natural products in the traditional sense and may retain more medicinal properties. The present invention obtains reared larvae similar to wild bat moth larvae by preparing a mixed culture matrix and monitoring and adjusting the environmental parameters in the culture chamber in real time.

[0042] (2) The present invention effectively removes potentially harmful microorganisms and reduces the number of pathogens by selecting a specific proportion of alpine meadow herbs, alpine shrub dwarf plants and alpine meadow mosses, sterilizing a portion of them and then inoculating with a fungal spore suspension, while retaining a portion of the basic structure of the original microbial community. This not only reduces the risk of pathogen infection, but also provides a better colonization space for the inoculated beneficial strains and optimizes the microbial ecological balance. Compared with the traditional artificial breeding method, the latter usually uses a single food source and fails to provide a complex ecological environment, resulting in the inability of certain specific bacterial communities in the larval intestine to colonize normally. The present invention significantly improves the diversity of microorganisms through the selection of mixed plant materials and moderate sterilization treatment. The optimized microbial ecological balance provides a better living environment for the larvae, improves the survival rate and growth rate, and further promotes the healthy growth of the larvae.

[0043] (3) The present invention ensures a high degree of consistency between the culture environment and the natural habitat by real-time monitoring of the environmental parameters of the natural habitat of the bat moth larvae, and adjusting the culture parameters in the culture chamber in real time according to the collected data. The real-time monitoring and adjustment mechanism enables the larvae to grow and develop under conditions close to natural conditions, reducing the stress response caused by environmental differences. Unlike the traditional artificial breeding environment, which is usually a constant state, this dynamic adjustment restores the natural environment to the greatest extent possible, improving the adaptability and health of the larvae. The stable growth environment not only promotes the rapid growth of the larvae, but also reduces the risk of pathogen infection, thereby increasing the overall survival rate. In addition, simulating the wild environment helps to study the interaction between the larvae and the environment, provides a data basis for the subsequent optimization of the culture method, and further promotes scientific research and technological development in related fields.

[0044] (4) The present invention combines the two features of constructing a mixed culture matrix and simulating a wild environment, producing a significant combined effect. The mixed culture matrix provides the necessary nutrients and a suitable microbial ecology, while the simulated wild environment ensures the consistency of the culture conditions with the natural habitat. The two complement each other and work together to promote the healthy growth of the larvae. By constructing a mixed culture matrix close to nature and simulating a wild environment, the present invention achieves a high degree of restoration of the growth environment of the bat moth larvae. This comprehensive measure not only improves the survival rate and growth rate of the larvae, but also ensures the diversity and stability of their intestinal microbial communities, ultimately achieving the goal of being as close to the wild state as possible. This highly restored culture condition provides reliable technical support for related research and applications, and has important scientific significance and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work.

[0046] Figure 1 The present invention is a flow chart of a method for bionic cultivation of bat moth larvae. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the specific embodiments disclosed below.

[0049] In the description of the present application, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the scope of protection of the present application. In addition, the terms "first", "second", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, features defined as "first", "second", etc. may explicitly or implicitly include one or more of the features. In the description of the invention, unless otherwise specified, "multiple" means two or more.

[0050] In the description of this application, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood by specific circumstances.

[0051] Exemplary Methods

[0052] like Figure 1 As shown, a bionic culture method for bat moth larvae comprises the following steps:

[0053] S1. Process biological samples from the intestines of wild bat moth larvae, extract target strains, and prepare fungal spore suspensions;

[0054] S2, washing, cutting and mixing leaves of alpine meadow herbaceous plants, young branches and leaves of alpine shrub dwarf plants and stems and leaves of alpine meadow mosses to obtain mixed plant materials;

[0055] S3, inoculating the fungal spore suspension onto the mixed plant material to form a mixed culture matrix;

[0056] S4, simulated wild environment;

[0057] S5, culturing the bat moth larvae in a mixed culture matrix;

[0058] Among them, the target strains include one or more of Penicillium, Aspergillus, Gibberellic acid and Trichoderma.

[0059] Below, each step will be described in detail.

[0060] Step S1 is to separate, purify and prepare a specific fungal strain from the intestine of wild bat moth larvae for subsequent bionic culture. The wild bat moth larvae are bat moth larvae living in a natural environment; the target strain refers to the fungal strain in the intestine of the wild bat moth larvae.

[0061] The processing of the biological sample from the intestine of wild bat moth larvae in step S1 includes:

[0062] S11, preparing potato dextrose agar medium;

[0063] S12, dissecting wild bat moth larvae, taking their intestinal contents, and inoculating them on potato dextrose agar medium;

[0064] S13, placing the inoculated culture medium in a constant temperature incubator, setting the temperature to 19-21°C, the humidity to 85%-90%, and culturing for 5-7 days;

[0065] S14. Pick a single colony and transfer it to a new potato dextrose agar medium for purification three or more times to obtain the target strain.

[0066] In a specific example, a potato dextrose agar (PDA) medium was prepared using a standard laboratory method, with 200 g of potato extract, 20 g of glucose, and 15 g of agar per 1000 ml of the medium, and a pH value of 6.0.

[0067] Furthermore, the head and tail of the bat moth larvae were tied tightly with thin threads, the body surface of the larvae was cleaned with sterile water and 75% alcohol, and the body surface was disinfected by soaking in 75% alcohol for 3 minutes; the larvae were dissected under sterile conditions, their intestines were taken out, and a sterile inoculation loop was used to inoculate them on PDA culture medium using the streak separation inoculation method.

[0068] Furthermore, the inoculated PDA culture medium was placed in a sterile constant temperature incubator, the temperature was set at 20°C, the humidity was set at 85%, and the incubator was kept in a dark environment for 5 days.

[0069] Furthermore, a single colony was picked from the culture medium, streaked onto a new PDA culture medium using a sterile inoculation loop, and streaking separation and purification were performed three times to obtain a purified target strain.

[0070] The method for preparing the fungal spore suspension in step S1 is: remove the mature fungal spores from the target strain, add sterile physiological saline, shake evenly, and adjust the spore concentration to 1×10 6 -10 8 / mL to obtain a fungal spore suspension.

[0071] In a specific embodiment, mature purified fungal spores of Cordyceps sinensis, Penicillium and Aspergillus on the culture medium were scraped off with a sterile scraper, transferred to a centrifuge tube containing sterile physiological saline, vortexed evenly with a vortex oscillator, and counted under a microscope using a hemocytometer, and the spore concentration was adjusted to 1×10 7 / mL, prepared into fungal spore suspension.

[0072] Through the above technical scheme, a high-purity fungal strain that fits the intestines of wild bat moth larvae is obtained, as well as a spore suspension with uniform dispersion and controllable concentration.

[0073] Step S2 selects plants common in the habitat of bat moth larvae and uses specific parts thereof to simulate the natural food source of the larvae.

[0074] The mixed plant material in step S2 contains 30-50% alpine meadow herbaceous plants, 20-40% alpine shrub dwarf plants and 20-40% alpine meadow mosses; wherein the alpine meadow herbaceous plants include one or more of Oxytropis purpurogena, Polygonum villosa and Pedullis dwarfis; the alpine shrub dwarf plants include one or more of Rhododendron microphylla, Potentilla fruticosa and Ceratoides cuspidatus; the alpine meadow mosses include one or more of Pseudocematophyte, Psoralea corylifolia and Black moss.

[0075] In a specific embodiment, leaves of Oxytropis purpurogena, Polygonum villosa, and Pedunculia dwarfum in the wild environment are collected; twigs and leaves of Rhododendron microphylla, Potentilla fruticosa, and Ceratoides cuspidatus; stems and leaves of Pseudo-thinwalled moss, sand moss, and black moss. The plant material is rinsed 3 times with sterile water to remove surface contaminants. The plant material is cut into 2 cm pieces using sterile scissors. The treated plant material is mixed in a ratio of 40% alpine meadow herbaceous plants, 30% alpine shrub dwarf plants, and 30% alpine meadow mosses.

[0076] Through the above technical scheme, it is ensured that the plant materials used are consistent with the natural food source of the bat moth larvae, and can provide basic nutrition and habitat, which is more conducive to the colonization of the target strain.

[0077] Step S2 also includes: sterilizing 20-60% of the mixed plant material;

[0078] In step S3, the fungal spore suspension is inoculated into the sterilized mixed plant material, and the inoculated mixed plant material is mixed with the remaining unsterilized mixed plant material to obtain a mixed culture matrix.

[0079] In a specific embodiment, 40% of the mixed plant material was sterilized by high pressure steam (121°C, 15 psi, 20 min); 1×10 7 / mL of fungal spore suspension is evenly sprayed onto the sterilized plant material, and the plant material is gently stirred to ensure that the fungal spores are evenly distributed; the inoculated plant material is mixed with the remaining unsterilized plant material to obtain a mixed culture matrix.

[0080] In another specific embodiment, non-sterile plant material is placed in a container containing a fungal spore suspension and soaked for 10 minutes. During the soaking process, ensure that the plant material is completely immersed in the suspension so that the spores are fully attached to the plant surface. The soaking time should not be too long to prevent the plant material from absorbing too much water, which will affect its physical structure and nutritional components. After the soaking is completed, the plant material is taken out and the excess water is gently drained.

[0081] Through the above technical solution, specific fungi are introduced into the culture matrix, and the beneficial microorganisms present in the plant material are retained to assist the colonization of the specific strains and maintain the balance of the larval intestinal microbial community. Among them, these beneficial microorganisms include but are not limited to plant endophytes and decomposing bacteria, which promote the colonization of specific strains in the larval intestine by producing metabolites or forming synergistic effects with specific strains, and help maintain the stability of the larval intestinal microecology.

[0082] Step S4 simulates the environmental parameters of the natural habitat of the bat moth larvae to provide the most suitable conditions for the growth and development of the larvae. The specific method includes:

[0083] S41. Real-time monitoring of environmental parameters in the natural habitat of bat moth larvae;

[0084] S42, adjusting the culture parameters in real time according to the environmental parameters so that the culture parameters are synchronized with the environmental parameters;

[0085] Both environmental parameters and culture parameters include temperature, humidity, light, air pressure, wind speed, rain, pH, carbon dioxide concentration, oxygen concentration and ultraviolet radiation.

[0086] In a specific embodiment, sensors are installed in the natural habitat of bat moth larvae to monitor environmental parameters such as temperature, humidity, light, air pressure, wind speed, rain, pH, carbon dioxide concentration, oxygen concentration and ultraviolet radiation in real time, with a monitoring frequency of once every 10 minutes.

[0087] Through the above technical scheme, a growth environment close to the natural habitat is provided for the bat moth larvae, which is beneficial to the growth and development of the larvae and provides the necessary conditions for further research on the interaction between the larvae and the environment.

[0088] Step S5 is to place the bat moth larvae in a mixed culture matrix and culture them in a simulated wild environment to achieve bionic culture of the larvae, which specifically includes:

[0089] S51, culturing the bat moth larvae in a mixed culture matrix at a larval density of 200-300 per square meter;

[0090] S52. During the culture process, the culture medium is replaced regularly, with a frequency of once every 3-5 days.

[0091] In a specific embodiment, the bat moth larvae are placed in a mixed culture matrix for culture, the larvae density is 250 per square meter, and the culture matrix is ​​replaced every 5 days during the culture process.

[0092] Furthermore, a 2 cm thick layer of soil or humus from the natural environment is spread on the bottom of the culture matrix. These substances usually contain rich fungal spores to help the fungi colonize in the intestines of the larvae.

[0093] Through the above technical scheme, the larvae are provided with suitable culture density, sufficient nutrition, clean environment and oxygen supply, thereby promoting the healthy growth of the larvae.

[0094] Further, step S6: biological monitoring of the cultured bat moth larvae to evaluate the culture effect and the health status of the larvae; specifically comprising:

[0095] monitoring the microbial composition of the larval gut;

[0096] The growth rate and mortality of the larvae were monitored, and the growth, development and health status of the larvae were recorded.

[0097] In a specific embodiment, the microbial composition in the larval intestine, especially the fungal colonization, is monitored every 7 days using microscopy or molecular biology techniques (ITS sequencing / qPCR).

[0098] Furthermore, the larval length was measured every 3 days, the larval mortality was recorded, and the hemolymphocyte count and phenoloxidase activity of the larvae were detected.

[0099] Through the above technical scheme, we can fully understand the growth and development of the larvae, monitor the changes in the intestinal microbiota, evaluate the effectiveness of the culture method, and provide data basis for the subsequent optimization of the culture method.

[0100] Exemplary Systems

[0101] A bat moth larvae bionic culture system, based on the above method, comprising:

[0102] Environmental parameter collection module, used to collect environmental parameters of the natural habitat of bat moth larvae;

[0103] A culture chamber for cultivating bat moth larvae;

[0104] The environmental parameter transmission module is connected to the environmental parameter acquisition module by signal, and is used to transmit the collected environmental parameters to the culture chamber control module;

[0105] A culture chamber environmental parameter monitoring module is used to monitor the culture parameters in the culture chamber;

[0106] The culture chamber control module is used to receive the environmental parameters transmitted by the environmental parameter transmission module and adjust the culture parameters of the culture chamber according to the collected environmental parameters.

[0107] Below, each module will be introduced in detail.

[0108] The environmental parameter collection module continuously and in real time monitors the environmental parameters of the natural habitat of bat moth larvae. It can comprehensively monitor environmental parameters such as temperature, humidity, light intensity, air pressure, wind speed, rainfall, pH, carbon dioxide concentration, oxygen concentration and ultraviolet radiation. The collected environmental parameters have high accuracy and stability.

[0109] In a specific embodiment, high-precision sensors are used, including temperature sensor (PT1000), humidity sensor (HIH6130), light sensor (TSL2561), air pressure sensor (BMP280), wind speed sensor (cup type or thermal type), pH sensor (PH4502C), carbon dioxide sensor (MG811), oxygen sensor (ME2-O2-Φ17) and ultraviolet sensor (GUVA-S12SD). The above sensors are integrated and installed in a waterproof and dustproof signal tower A, and the signal tower A integrating multiple sensors is installed in the natural habitat of bat moth larvae on the Qinghai-Tibet Plateau.

[0110] The environmental parameter transmission module transmits the data monitored in real time by the environmental parameter acquisition module to the culture chamber control module in a stable and reliable manner to ensure the real-time and accuracy of data transmission; it uses wireless transmission technology to avoid the limitations brought by wired transmission and adapt to the distance between the field environment and the laboratory environment.

[0111] In a specific embodiment, a signal tower B is installed at the laboratory location, and the data monitored by the signal tower A on the Qinghai-Tibet Plateau is transmitted in real time to the signal tower B located in the laboratory through the 5G network or satellite communication. The 5G network or satellite communication technology is used to ensure the stability and speed of data transmission, and the data transmission frequency is once every 10 minutes to ensure the timeliness and accuracy of the data.

[0112] The culture chamber can adjust the environmental parameters in the culture chamber according to the instructions of the culture chamber control module, realize environmental simulation, and provide a controllable culture space for bat moth larvae.

[0113] The incubation chamber environmental parameter monitoring module monitors the temperature, humidity, light intensity, gas concentration, ultraviolet radiation and other parameters in the incubation chamber in real time to ensure the stability of the incubation environment, and feeds back the monitored data to the incubation chamber control module for real-time adjustment.

[0114] In a specific embodiment, multiple sensors are installed inside the culture chamber to monitor parameters such as temperature, humidity, and light intensity in real time, and the data is fed back to the culture chamber control module in real time to verify the consistency of the culture chamber environmental parameters with the natural environment, and automatically adjust the culture chamber environmental parameters based on the feedback information.

[0115] The culture chamber control module receives the environmental parameter data from the environmental parameter transmission module and the culture parameter data from the culture chamber environmental parameter monitoring module, processes and analyzes the received data, and issues instructions to adjust the environmental parameters in the culture chamber according to the preset parameter range.

[0116] The culture chamber control module includes an automatic control system based on a PLC (programmable logic controller), and actuators connected to the control system including a thermostat, a humidifier / dehumidifier, an LED lamp, a gas flow controller, a small fan and an ultraviolet lamp.

[0117] In a specific embodiment, after the culture chamber control module receives the data from the signal tower B, it first analyzes the data and then issues instructions based on a preset parameter range to control the thermostat, humidifier / dehumidifier, LED light, gas flow controller, small fan and other actuators in the culture chamber to adjust the temperature, humidity, light, gas concentration, wind speed and other parameters in the culture chamber in real time to ensure that the environmental parameters in the culture chamber are synchronized with the natural environment.

[0118] By using the system composed of the above modules, fine control of the culture environment of bat moth larvae is achieved, which can highly restore the natural habitat of the larvae and provide a guarantee for the bionic culture of bat moth larvae.

[0119] Unless otherwise specified in the following examples, all raw materials are commercially available or prepared by conventional methods in the art.

[0120] Wild bat moth larvae were collected from the Qiangtang National Nature Reserve in Nagqu, Tibet in the summer of 2023 and provided by the Kunming Institute of Zoology, Chinese Academy of Sciences, number WTC-2023-QZ01, which specializes in biodiversity conservation and wildlife research and has a legal wild collection permit. These larvae come from natural environments, and their intestinal microbial communities have not been interfered with by humans, maintaining their most original state.

[0121] Artificially reared bat moth larvae, from a Beijing-based biotechnology company, batch BJBT-BF2024-H01, are reared on a large scale according to commercial standards to provide high-quality and consistent experimental materials.

[0122] Example 1

[0123] A bionic culture method for bat moth larvae comprises the following steps:

[0124] S1. Prepare potato dextrose agar medium; dissect wild bat moth larvae, take out their intestinal contents, and inoculate them on potato dextrose agar medium; place the inoculated medium in a constant temperature incubator, set the temperature to 20°C and the humidity to 85%, and culture for 5 days; pick single colonies of Penicillium, Aspergillus, Gibberellum and Trichoderma, transfer them to new potato dextrose agar medium for purification three times to obtain Penicillium, Aspergillus, Gibberellum and Trichoderma; use a sterile scraper to scrape off the mature and purified fungal spores of Penicillium, Aspergillus, Gibberellum and Trichoderma on the medium, transfer them to a centrifuge tube containing sterile 0.9% NaCl saline, use a vortex oscillator to oscillate evenly, use a hemocytometer to count under a microscope, and adjust the spore concentration to 1×10 7 / mL to obtain a fungal spore suspension.

[0125] S2. Mix the same mass of leaves of Oxytropis purpurogena, Polygonum villosa and Pedunculaceae to obtain alpine meadow herbaceous plant materials; mix the same mass of Rhododendron microphylla, Potentilla fruticosa and Ceratoides cuspidatum to obtain alpine shrub dwarf plant materials; mix the same mass of Pseudo-thinwalla, Psoralea corylifolia and Black moss to obtain alpine meadow moss plant materials; rinse the alpine meadow herbaceous plant materials, alpine shrub dwarf plant materials and alpine meadow moss plant materials with sterile water for 3 times, remove surface contaminants, cut them into 2 cm pieces with sterile scissors, and mix them in a mass ratio of 4:3:3 to obtain mixed plant materials.

[0126] S3, 20% of the mixed plant materials were sterilized by high pressure steam (121°C, 15 psi, 20 min); 1×10 7 / mL of fungal spore suspension is evenly sprayed onto the sterilized plant material, and the plant material is gently stirred to ensure that the fungal spores are evenly distributed; the inoculated plant material is mixed with the remaining unsterilized plant material to obtain a mixed culture matrix.

[0127] S4. Real-time monitoring of environmental parameters of the natural habitat of bat moth larvae, and real-time adjustment of the cultivation parameters of the bat moth bionic cultivation, so that the cultivation parameters are synchronized with the environmental parameters; wherein the environmental parameters and the cultivation parameters both include temperature, humidity, light, air pressure, wind speed, rain, pH, carbon dioxide concentration, oxygen concentration and ultraviolet radiation;

[0128] S5. The bat moth larvae were cultured in a mixed culture matrix with a larval density of 250 per square meter. The culture matrix was replaced every 5 days during the culture process.

[0129] Example 2

[0130] Different from Example 1, in step S3 of this example, 40% of the mixed plant materials are sterilized by high-pressure steam.

[0131] Example 3

[0132] Different from Example 1, in step S3 of this example, 60% of the mixed plant materials are sterilized by high-pressure steam.

[0133] Example 4

[0134] Different from Example 1, in step S3 of this example, 80% of the mixed plant materials are sterilized by high-pressure steam.

[0135] Experimental Example 1

[0136] 40 4-5 instar bat moth larvae obtained by the bionic culture method of Examples 1-4, 4-5 instar wild bat moth larvae collected from the wild, and 4-5 instar bat moth larvae raised artificially were selected;

[0137] Tie the head and tail of the bat moth larvae with a thin thread, clean the larvae with sterile water and 75% alcohol, and soak them in 75% alcohol for 3 minutes to disinfect the body surface; dissect the larvae under sterile conditions, take out the intestines, rinse them with 0.01M PBS buffer, add 1mL of sterile water to homogenize, and dilute the homogenate in a 10-fold gradient to 10 -1 -10 -8 .

[0138] Take 10 -1 -10 -8 0.1mL of each dilution solution was spread and inoculated. Three plates were inoculated with each culture medium at each dilution. The spread plates were placed in a temperature gradient of 10℃, 15℃, 20℃, and 25℃ for 5 days. The growth of the colonies was observed and counted using the countability principle. The typical target colonies on each plate were counted and separated and purified. The results are shown in the following table:

[0139] Table 1 Detection rate results

[0140]

[0141]

[0142] Comparing the data under wild and artificial breeding conditions, it can be clearly seen that the detection rate of many bacterial species (especially fungi) in artificial breeding environments is extremely low or even zero. This is because artificial breeding in the prior art usually fails to provide a complex ecological environment, especially in terms of microbial ecology. Simple breeding methods fail to simulate the microbial diversity existing in the natural environment, resulting in the inability of certain specific bacterial communities in the larval intestine to colonize normally.

[0143] The detection rates of most strains in Examples 1-4 are close to those in the wild. This is because, by selecting plant materials common in the habitats of bat moth larvae and using specific parts thereof to simulate the natural food sources of the larvae, these plant materials not only provide the necessary nutrients, but also retain the microbial communities on their surface and inside, including endophytes and decomposers. These microorganisms promote the colonization of specific strains in the larval intestine and help maintain the stability of the larval intestinal microecology. Although sterilization removes both beneficial and harmful microorganisms, key beneficial strains are reintroduced in subsequent steps by inoculating specific fungal spore suspensions. This allows the culture environment to be closer to the natural state, so that the overall performance of Examples 1-4 can fit the wild state.

[0144] In Examples 1-4, the detection rate of most species reached a peak in Example 2 (40% sterilization treatment), and then gradually decreased with the increase of the sterilization ratio. For example, cold seawater Flavobacterium, oxidative Microbacterium, and Tsukamurella pulmonaria achieved a high detection rate in Example 2, but significantly decreased in Examples 3 and 4. Some high-stability bacterial communities (such as malt-flavored carnibacterium and anti-tumor active marine bacteria) maintained a relatively stable high detection rate throughout the process. This is because the 40% sterilization treatment effectively reduces the number of competitive microorganisms and provides better colonization space for the inoculated beneficial strains. At the same time, this sterilization ratio does not excessively destroy the physical and chemical properties of the plant material, maintains its quality as a food source, and is conducive to the colonization of specific strains. When the sterilization ratio exceeds 40%, especially when it reaches 60% and 80%, excessive sterilization will destroy all microbial ecology on the plant material, including the original beneficial microbial basis. This not only affects the colonization efficiency of the inoculated strain, but also changes the physical and chemical properties of the plant material itself, indirectly affects the quality of the food source of the larvae, and further reduces the detection rate of specific strains.

[0145] Experimental Example 2

[0146] Based on the bionic culture method for bat moth larvae provided in Examples 1-4, the survival rate of 200 bat moth larvae from inoculation to 4-5 instars in each example was counted; during the culture process, the length of the larvae was measured every 3 days, and the growth rate of the larvae was calculated. The results are shown in the following table:

[0147] Table 2 Survival rate and growth rate results

[0148] Example 1 Example 2 Example 3 Example 4 Survival rate (%) 85 88 83 79 Growth rate (mm / d) 0.5 0.52 0.48 0.45

[0149] The survival rate was 85% at 20% sterilization, rose to a maximum of 88% at 40% sterilization, and then gradually decreased to 83% and 79% with further increase in sterilization ratio (60% and 80%). Similarly, the growth rate was 0.5 mm / d at 20% sterilization, reached a maximum of 0.52 mm / d at 40% sterilization, and then decreased to 0.48 mm / d and 0.45 mm / d with increase in sterilization ratio.

[0150] This is because different sterilization ratios have an impact on all microorganisms on plant materials, including beneficial and harmful microorganisms. Under the 40% sterilization treatment conditions, although most of the microorganisms were removed, specific beneficial strains were successfully introduced by inoculating fungal spore suspensions. This treatment method not only reduces the potential pathogens in the original microbial community, but also provides a better colonization environment for the inoculated beneficial strains. At the same time, some of the unsterilized plant materials retain a certain natural microbial basis, which helps to maintain the diversity and stability of the larval intestinal microbial community. When the sterilization ratio exceeds 40%, especially when it reaches 60% and 80%, excessive sterilization not only removes more microorganisms, but also destroys the physical and chemical properties of the plant materials and changes its quality as a food source. This not only affects the colonization efficiency of the inoculated strains, but also indirectly affects the health and growth rate of the larvae. Excessive sterilization ratios lead to a reduction in microbial diversity, forming a microenvironment that is not conducive to the colonization of specific strains, thereby reducing the survival rate and growth rate of the larvae. While removing most of the microorganisms, the sterilization treatment of about 40% retains the original structure and nutrients of some plant materials, maintaining its quality as a food source. This ratio of sterilization optimizes the cleanliness of the culture matrix while restoring the necessary beneficial microorganisms through inoculation, providing better living and growth conditions for the larvae.

[0151] Taking all the above factors into consideration, a sterilization ratio of about 40% (i.e., Example 2) is an ideal bionic culture condition. Within this range, although sterilization will remove all microorganisms, the key beneficial strains are successfully restored through subsequent inoculation of fungal spore suspension. At this ratio, the culture matrix is ​​not only moderately clean, but also provides a good colonization environment for the inoculated beneficial microorganisms, simulating the wild environment to the maximum extent, which is conducive to the healthy growth of bat moth larvae.

[0152] The above is based on the ideal embodiment of the present invention. Through the above description, relevant personnel can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present invention is not limited to the content in the specification, and the technical scope must be determined according to the scope of the claims.

Claims

1. A bionic culture method for bat moth larvae, characterized in that: The following steps are involved: S1. Process biological samples from the intestines of wild bat moth larvae, extract target strains, and prepare fungal spore suspensions; S2, washing, cutting and mixing leaves of alpine meadow herbaceous plants, young branches and leaves of alpine shrub dwarf plants and stems and leaves of alpine meadow mosses to obtain mixed plant materials; S3, inoculating the fungal spore suspension onto the mixed plant material to form a mixed culture matrix; S4, simulated wild environment; S5, culturing the bat moth larvae in a mixed culture matrix; Wherein, the target strains include one or more of Penicillium, Aspergillus, Gibberellic acid and Trichoderma.

2. The bionic culture method of bat moth larvae according to claim 1, characterized in that: The processing of the biological sample from the intestine of wild bat moth larvae in step S1 includes: S11, preparing potato dextrose agar medium; S12, dissecting wild bat moth larvae, taking their intestinal contents, and inoculating them on potato dextrose agar medium; S13, placing the inoculated culture medium in a constant temperature incubator, setting the temperature to 19-21°C, the humidity to 85%-90%, and culturing for 5-7 days; S14. Pick a single colony and transfer it to a new potato dextrose agar medium for purification three or more times to obtain the target strain.

3. The bionic culture method of bat moth larvae according to claim 1, characterized in that: The method for preparing the fungal spore suspension in step S1 is: removing mature fungal spores from the target strain, adding sterile physiological saline, shaking evenly, and adjusting the spore concentration to 1×10 6 -10 8 / mL to obtain a fungal spore suspension.

4. The bionic culture method of bat moth larvae according to claim 1, characterized in that: The mixed plant materials in step S2 contain 30-50% alpine meadow herbaceous plants, 20-40% alpine shrub dwarf plants and 20-40% alpine meadow mosses; wherein the alpine meadow herbaceous plants include one or more of Oxytropis purpurogena, Polygonum villosa and Pedulex dwarfus; the alpine shrub dwarf plants include one or more of Rhododendron microphylla, Potentilla fruticosa and Ceratoides cuspidatus; and the alpine meadow mosses include one or more of Pseudocematophyte, Psoralea corylifolia and Black moss.

5. The bionic culture method of bat moth larvae according to claim 1, characterized in that: The step S2 further comprises: sterilizing 20-60% of the mixed plant material; In step S3, the fungal spore suspension is inoculated into the sterilized mixed plant material, and the inoculated mixed plant material is mixed with the remaining unsterilized plant material to obtain a mixed culture matrix.

6. The bionic culture method of bat moth larvae according to claim 1, characterized in that: The method for simulating a wild environment in step S4 includes: S41. Real-time monitoring of environmental parameters in the natural habitat of bat moth larvae; S42, adjusting the culture parameters in real time according to the environmental parameters to synchronize the culture parameters with the environmental parameters; The environmental parameters and culture parameters include temperature, humidity, light, air pressure, wind speed, rain, pH, carbon dioxide concentration, oxygen concentration and ultraviolet radiation.

7. The bionic culture method of bat moth larvae according to claim 1, characterized in that: The step S5 comprises: S51, culturing the bat moth larvae in a mixed culture matrix at a larval density of 200-300 per square meter; S52. During the culture process, the culture medium is replaced regularly, with a frequency of once every 3-5 days.

8. The bionic culture method of bat moth larvae according to claim 1, characterized in that: The step S6 is also included: monitoring the microbial composition of the larval gut; The growth rate and mortality of the larvae were monitored, and the growth, development and health status of the larvae were recorded.

9. A bionic culture system for bat moth larvae, based on the method according to any one of claims 1 to 8, characterized in that: include; Environmental parameter collection module, used to collect environmental parameters of the natural habitat of bat moth larvae; A culture chamber for cultivating bat moth larvae; An environmental parameter transmission module, connected to the environmental parameter acquisition module by signal, and used to transmit the collected environmental parameters to the culture chamber control module; A culture chamber environmental parameter monitoring module is used to monitor the culture parameters in the culture chamber; The culture chamber control module is used to receive the environmental parameters transmitted by the environmental parameter transmission module and adjust the culture parameters of the culture chamber according to the collected environmental parameters.

10. The bionic culture system for bat moth larvae according to claim 9, characterized in that: The environmental parameter transmission module includes a signal tower A and a signal tower B connected by signals; The environmental parameter acquisition module includes a temperature sensor, a humidity sensor, a light sensor, an air pressure sensor, a wind speed sensor, a pH sensor, a carbon dioxide sensor, an oxygen sensor and an ultraviolet sensor integrated in the signal tower A; The culture chamber control module includes an automatic control system based on a programmable logic controller, and an actuator connected to the control system including a thermostat, a humidifier, a dehumidifier, an LED lamp, a gas flow controller, a small fan and an ultraviolet lamp.

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