Compound refrigeration method for inhibiting development of silkworm chrysalis of ricinus communis

Abstract

The invention discloses a compound refrigeration method for inhibiting development of silkworm chrysalis of ricinus communis, belongs to the technical field of insect development regulation and long-term preservation, and solves the problems of failure of eclosion after recovery or unhealthy adults caused by irreversible physiological injury to pupa bodies in a traditional single long-term refrigeration method. According to the scheme, the method comprises the following steps: starting from the fifth age of castor silkworms, slowly reducing the raising temperature from 25-28 DEG C to 20 DEG C or below, and carrying out first low-temperature preservation after the castor silkworms pupate; then transferring to a first recovery environment for recovery culture until the pupa body develops to a predetermined stage; carrying out secondary low-temperature storage; and finally, completing eclosion culture in a controlled final recovery environment. According to the method, a recovery period is set between two times of low-temperature preservation, and the pupa body is subjected to necessary physiological restoration, so that the physiological function of the pupa body is maintained while long-term development inhibition is realized, and the method is mainly used for long-term preservation of castor silkworm chrysalis and ensures healthy and high-success-rate eclosion after recovery of the castor silkworm chrysalis.

Description

Technical Field

[0001] This invention belongs to the field of insect development regulation and long-term preservation technology, specifically relating to a double-layer cold storage method for inhibiting the development of castor silkworm pupae. Background Technology

[0002] Currently, the long-term preservation of castor silkworm pupae often employs a single method of long-term cryopreservation to inhibit their development. While this method can extend the preservation time to some extent, the effective preservation period is short, typically within two months. Prolonged preservation can cause irreversible physiological damage to the pupae, leading to failed emergence or unhealthy adults after revival. The main reason for this problem is that while prolonged low temperatures can effectively inhibit development, they also gradually weaken the pupae's basic physiological activity, disrupting their normal metabolic balance and energy conversion processes. The difficulty in solving this problem lies in balancing the contradiction between developmental inhibition and the maintenance of physiological activity: simply shortening the cryopreservation time fails to achieve long-term inhibition, while excessively extending the cryopreservation time exacerbates the risk of physiological damage. Therefore, finding a preservation method that can simultaneously inhibit development over a long period and ensure the healthy emergence of the pupae presents practical challenges. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0004] Another objective of this invention is to provide a double-layer cold storage method for inhibiting the development of castor silkworm pupae, which can effectively inhibit the development of castor silkworm pupae for a long time while maintaining the physiological functions of the pupae, ensuring that they can complete their eclosion healthily and with a high success rate after final recovery.

[0005] To achieve these objectives and other advantages of the present invention, a double-refrigeration method for inhibiting the development of castor silkworm pupae is provided, comprising the following steps: S1: Starting from the fifth instar of the castor silkworm, gradually reduce the rearing temperature from 25~28℃ to below 20℃, so that the castor silkworm can complete the maturation and pupation in a low temperature environment; 12 hours after the castor silkworm pupates, place it in the first low temperature environment for the first low temperature preservation, the preservation temperature is 4~8℃, and the preservation time is 60~70 days. S2: After the first low-temperature preservation is completed, the castor silkworm pupae are transferred to the first recovery environment for the first recovery culture. The temperature of the first recovery environment is 24~28℃ and the recovery time is 12h until the pupae develop to the predetermined recovery stage. In the first recovery environment, the relative humidity is controlled at 70%~80%, and air circulation is maintained at an airflow speed of 0.1~0.3 m / s; during the first recovery culture, 10~12 hours of light is provided, with a light intensity of 50~150 lux. S3: After the first recovery culture is completed, the castor silkworm pupae are placed in a second low-temperature environment for a second low-temperature preservation at a temperature of 4~8℃ for 30~40 days. S4: After completing the second cryopreservation, the castor silkworm pupae are transferred to the final recovery environment for emergence culture until they complete emergence; The final recovery environment temperature is 25~28℃. Starting from the 24th hour of the final recovery environment, a 12-hour light cycle is provided every day, and the light intensity is controlled at 100~200 lux. During the low-temperature storage period, environmental checks and ventilation are carried out every 12 to 18 days in steps S1 and S3, with a ventilation time of 10 to 20 minutes. During this period, the temperature fluctuation of the low-temperature environment should not exceed ±1℃.

[0006] Traditional long-term cryopreservation methods can easily cause irreversible physiological damage to the pupae during prolonged suppression of castor silkworm pupal development, leading to failed emergence or poor adult health after recovery. This invention divides the long-term cryopreservation process into a phased cycle of "first cryopreservation – recovery culture – second cryopreservation – final emergence." Starting from the fifth instar of the castor silkworm, the rearing temperature is gradually lowered from 25-28℃ to below 20℃, allowing the silkworms to mature and pupate in a low-temperature environment. After the first cryopreservation induces dormancy in the pupae, a recovery culture stage with suitable temperature, humidity, light, and airflow conditions is inserted, enabling the pupae to undergo necessary physiological repair and energy conversion, laying the foundation for withstanding subsequent cryopreservation and maintaining vitality. This invention, through the organic combination of phased cryopreservation and critical recovery periods, achieves both long-term developmental inhibition and effective maintenance of pupal physiological functions, ultimately ensuring that the pupae can complete the emergence process healthily and with a high success rate after recovery.

[0007] Preferably, the criterion for determining when the pupae have developed to the predetermined recovery stage in step S2 is that more than 80% of the pupae in the group change from the light yellow color of the early pupal stage to a uniform yellowish-brown color.

[0008] This invention is based on insect developmental morphology, specifically the fact that changes in the body color of the pupa are a reliable external representation of its internal physiological development stages. The light yellow color in the early pupal stage indicates that the epidermis is not yet fully ossified and pigment deposition is incomplete, while the transformation to a uniform yellowish-brown signifies that the pupa has completed a specific epidermal tanning process and some organ differentiation, reaching a physiologically stable "preparation stage" capable of withstanding subsequent low-temperature stress. This invention uses "more than 80% of the pupae in the group completing this color change" as a statistically verifiable and repeatable objective criterion. This method transforms a subjective biological development process into a clear operational indicator, effectively improving the operability and consistency of the method. It ensures that each batch of pupae can be transferred to a second cryopreservation at the optimal physiological point, providing a crucial guarantee for the health of subsequent emergence.

[0009] Preferably, during the first recovery culture process in step S2, after the pupa's body color reaches the predetermined recovery stage and before proceeding to step S3, a stabilization transition step is also included: the temperature of the first recovery environment is reduced from 24-28°C to 15-18°C at a rate of 0.5-1°C per hour, and maintained at 15-18°C for 6-12 hours before being transferred to the second low-temperature environment.

[0010] When transitioning directly from a warm recovery environment to a low-temperature refrigerated environment, the sudden drop in temperature can trigger a "cold shock" phenomenon. This drastic temperature change can damage the cell membrane structure of the pupa, leading to a sharp decrease in enzyme activity and metabolic disorders, thus causing potential harm and affecting subsequent survival rates. This invention designs a stable transition step, gradually reducing the ambient temperature from 24-28°C to 15-18°C at a slow rate of 0.5-1°C per hour, and maintaining this intermediate temperature for 6-12 hours. This allows the pupa's physiological functions, such as membrane lipid composition, metabolic rate, and the synthesis of cold-resistant substances, to gradually adapt and adjust. This gentle cooling mode effectively stimulates the pupa's internal protective mechanisms, enabling a smooth transition from an active state to a dormant state, thereby avoiding cold shock damage and improving the survival rate after the second refrigeration and the health of the subsequently emerging adults.

[0011] Preferably, a stepped heating method is used in the final recovery culture process of step S4: First, place the pupae that have just been taken out of the second low temperature environment at 18~20℃ for 8~12 hours; Subsequently, the temperature was increased to 20-25°C at a rate of 0.5-1°C per hour and maintained for 12-16 hours; Finally, raise the temperature to 25-28°C at a rate of 0.5-1°C per hour and maintain it until the molting is complete.

[0012] When pupae that have been refrigerated for a long time are directly introduced into a higher-temperature emergence environment from a dormant state, a "heat shock" phenomenon may occur due to the rapid temperature rise. This phenomenon can lead to abnormal metabolic bursts, protein denaturation, and disordered developmental programs, resulting in deformed emergence or emergence failure. This invention designs a step-by-step heating method. First, pupae that have just been taken out of a low-temperature environment are placed at 18-20°C for 8-12 hours to allow them to restore basic metabolic functions. Then, the temperature is raised to 20-25°C at a rate of 0.5-1°C per hour and maintained for 12-16 hours to further activate their physiological systems. Finally, the temperature is raised to the final emergence temperature of 25-28°C at the same slow rate. This controlled warming process simulates the mild diurnal temperature variation in nature, which gradually "awakens" the pupa's metabolic, nervous, and endocrine systems. Hormones that control eclosion are secreted in an orderly manner, and the development and reorganization of cells and organs proceed synchronously. This effectively avoids heat shock damage, ensures the smooth completion of eclosion, a key life activity, and ultimately improves the eclosion success rate and the health and vitality of adult insects.

[0013] Preferably, during the first recovery culture in step S2, a water replenishment treatment is performed: water is atomized into droplets with a diameter of 5-15 micrometers using an atomizing device and sprayed evenly on the surface of the pupa. The relative humidity of the treatment environment is maintained at 90-95%, and each treatment lasts for 10-15 minutes. After treatment, the relative humidity is restored to the normal relative humidity of the first recovery environment of 70%-80%.

[0014] During the initial recovery culture process, pupae face the risk of physiological dehydration due to prolonged low-temperature storage. While maintaining conventional environmental humidity can prevent further water loss, it is difficult to actively and efficiently replenish the water lost during the initial storage, potentially limiting the physiological repair process and affecting tolerance to subsequent second-stage cold storage. This invention addresses this by actively and controlledly replenishing surface moisture. Atomizing water into fine droplets of 5-15 micrometers in diameter using an atomizing device and spraying them evenly onto the pupal surface, while simultaneously maintaining a temporary relative humidity of 90-95% for 10-15 minutes, this method leverages the ease with which the fine water mist is absorbed by the pupal surface. Combined with a temporary near-saturated humidity environment, it maximizes water penetration. Subsequent restoration of normal humidity avoids the potential risks associated with prolonged high humidity. This moisture replenishment treatment effectively alleviates the physiological dehydration of pupae, promotes the recovery of cellular metabolic activity and internal physiological functions, and lays the necessary physiological foundation for the pupae to successfully survive subsequent cold storage and emerge as adults.

[0015] Preferably, within 2 hours after the completion of the moisture replenishment treatment and restoration of normal humidity control, a periodic micro-airflow circulation is initiated and executed, which is performed for 2 complete cycles. Each complete cycle lasts for 60 minutes, specifically: for the first 50 minutes, the airflow speed is maintained at the normal flow rate of the first restored environment of 0.1~0.3 m / s, and then for the next 10 minutes, the airflow speed is adjusted to a high-speed airflow of 0.8~1.2 m / s.

[0016] After water replenishment, a stagnant air boundary layer tends to form around the pupae, leading to insufficient oxygen supply, carbon dioxide accumulation, and uneven temperature and humidity distribution within the microenvironment. This may affect the pupae's respiratory metabolism and their ability to fully absorb and utilize water. This invention addresses this by introducing a controlled, periodic micro-airflow circulation after water replenishment. Specifically, two cycles are initiated within 2 hours of restoring normal humidity. For the first 50 minutes of each 60-minute cycle, a normal low-speed airflow (0.1–0.3 m / s) is maintained to ensure environmental stability, while the airflow speed is significantly increased to 0.8–1.2 m / s for the following 10 minutes. This method effectively disturbs and breaks up the stagnant air boundary layer around the pupae using short-duration, high-velocity airflow, forcing air exchange and renewal. This increases local oxygen concentration, disperses accumulated carbon dioxide, and makes the temperature, humidity, and gas composition of the microenvironment more uniform. This periodic airflow circulation significantly improves the gas exchange efficiency and uniformity of the recovery environment without significantly reducing the pupae's surface humidity and affecting their water absorption, creating a more stable and efficient gaseous environment for the pupae's physiological recovery.

[0017] Preferably, periodic micro-airflow circulation is performed during the first restoration of the environment under illumination; the operation of water replenishment is performed during the dark phase; during the periodic micro-airflow circulation after water replenishment and restoration of normal relative humidity control, the airflow velocity of the periodic micro-airflow circulation is adjusted from 0.8~1.2m / s to 0.4~0.5m / s, and the duration is 20min.

[0018] During the dark phase of recovery culture, the periodic high-speed airflow circulation generated by the arrhythmic mechanical vibrations and noise may act as an abnormal environmental signal, interfering with the pupal's internal biological clock and disrupting its normal physiological metabolic rhythm recovery process during the dark resting period. This could increase physiological stress and potentially affect the quality of recovery. This invention addresses this by matching the intensity of physical disturbance at different times with the physiological state of the pupal. Specifically, during the light phase, periodic micro-airflow circulation is performed as originally planned. However, during the dark phase, the airflow pattern after water replenishment is adjusted, replacing the 10-minute high-velocity (0.8–1.2 m / s) airflow with a medium-velocity airflow that lasts for 20 minutes and remains constant at 0.4–0.5 m / s. This differentiated regulation ensures that during the dark resting period, the necessary microenvironment homogenization and gas exchange are maintained through a continuous and stable medium-velocity airflow, while minimizing the physical disturbance caused by high-intensity, short-pulse airflow that interferes with the pupal's resting state. This approach effectively reduces the negative impact on the pupal's internal biological clock and physiological rhythms. While ensuring environmental uniformity, it maintains the stability of the rhythmic recovery of the pupal's physiological metabolism (such as respiratory metabolism and hormone secretion), thereby improving the overall recovery culture effect.

[0019] Preferably, the specific implementation of transferring the castor silkworm pupae to the final recovery environment for eclosion culture in step S4 is as follows: placing the pupae in a single layer on an eclosion frame with a three-dimensional support structure. The eclosion frame is composed of multiple parallel grid plates with a vertical distance of 8-12 cm between layers. The surface of the grid plates is made of an inert material with a roughness Ra value between 3.2 and 12.5 micrometers. When placing the pupae, ensure that there is a distance between adjacent pupae that is at least 1.5 to 2 times their maximum body diameter. During the eclosion culture period, rotate the entire eclosion frame 90 to 180 degrees every 48 to 72 hours.

[0020] In the final recovery environment, pupae subjected to prolonged double-layer refrigeration suffer from uneven local microenvironmental conditions such as light and airflow due to their fixed location, leading to developmental defects such as asynchronous emergence, decreased emergence rate, and incomplete wing extension in adults. This invention eliminates the microenvironmental differences caused by static location by optimizing the physical layout and environmental exposure of pupae. Specifically, pupae are placed in a single layer on a three-dimensional emergence frame composed of multiple parallel grid plates, ensuring that adjacent pupae maintain a distance of at least 1.5 to 2 times their maximum body diameter. The grid plate surface has an inert material with a specific roughness between 3.2 and 12.5 micrometers. Furthermore, during the emergence cultivation period, the emergence frame is rotated 90 to 180 degrees every 48 to 72 hours. This arrangement avoids mutual interference and disease transmission among pupae by placing them in a single layer and maintaining spacing. The three-dimensional grid structure provides necessary support points for the emergence process, and its surface roughness prevents pupae from sliding while facilitating adult grasping. Regular orientation adjustments ensure that all pupae receive equal light, temperature, and airflow stimulation periodically, thereby guaranteeing the synchronization of endocrine signals and the normal completion of emergence behavior. In particular, it promotes the full extension and normal shaping of adult wings under the action of gravity, ultimately effectively improving the synchronicity of emergence, success rate, and health quality of adults.

[0021] Preferably, the mesh plate of the molting frame is provided with multiple shallow grooves that match the shape of the pupa. Each shallow groove is used to fix one pupa in place. The depth of the shallow groove is 30% to 50% of the average height of the pupa, and the inner surface roughness Ra value of the shallow groove is maintained in the range of 3.2 to 12.5 micrometers. The shallow grooves are distributed in an array on the mesh plate. The center distance between adjacent shallow grooves is equal to 1.5 to 2 times the distance between adjacent pupae. The opening size of the shallow groove is larger than the maximum body diameter of the pupa to ensure that the pupa is stably embedded without being completely enclosed.

[0022] During the periodic rotational feathering process, the pupa may slide, roll, or shift position due to physical disturbances, causing it to collide with the grid plate, change orientation, or even fall off, thus interfering with the stable feathering process and affecting the final success rate. This invention addresses this by creating an array of shallow grooves on a grid plate that matches the shape of the pupa. Each groove has a depth of 30%–50% of the average height of the pupa, maintains an inner surface roughness of 3.2–12.5 micrometers (Ra value), and the center-to-center distance between adjacent grooves is 1.5–2 times the distance between the pupa segments. Simultaneously, the groove opening size is ensured to be larger than the maximum diameter of the pupa. This invention utilizes the groove structure to provide precise local positioning and lateral support for the pupa. The depth limitation and rough inner surface significantly increase the static friction and fit between the pupa and the grid plate, effectively resisting external forces from the rotational operation. The opening size design ensures that while the pupa is stably embedded, its exposed portion still maintains necessary gas exchange and environmental sensing capabilities. This design significantly reduces the risk of pupal displacement during dynamic environmental adjustments, ensuring the normal progress of molting and thus improving the stability and success rate of the overall molting process.

[0023] Preferably, in step S1, the temperature is reduced from 25 to 28°C to below 20°C at a rate of 0.5 to 1.5°C / d.

[0024] During the cooling induction stage before pupation of castor silkworms, a rapid temperature drop may cause developmental stress or stagnation, while a slow drop may fail to effectively induce them into a low-temperature adaptation state, thus affecting the uniformity of pupation and the initial quality of the pupae. This invention quantifies the process of "gradually reducing the rearing temperature" by lowering the temperature at a specific rate of 0.5~1.5℃ per day. This method simulates the mild temperature changes during seasonal transitions in nature, allowing castor silkworms in their fifth instar to calmly adjust their metabolic rate and physiological functions, smoothly transitioning from a normal temperature growth state to a low-temperature induction state. This controlled cooling mode effectively avoids the physiological shock caused by sudden temperature changes, ensuring that the maturation and pupation processes are completed synchronously and smoothly in a low-temperature environment. This lays an important foundation for obtaining pupae with consistent development and healthy physiological foundations, as well as for the successful subsequent double-layer cold storage.

[0025] The present invention has at least the following beneficial effects: 1. Starting from the fifth instar of the castor silkworm, this invention gradually lowers the rearing temperature from 25-28℃ to below 20℃, allowing the castor silkworms to complete maturation and pupation in a low-temperature environment. Through a dual cold storage cycle of "low-temperature dormancy - recovery development - low-temperature dormancy again - final emergence," the contradiction between long-term developmental inhibition and the maintenance of pupal physiological activity is effectively balanced. While achieving a cumulative preservation period of approximately 90-110 days, this invention significantly improves the success rate of emergence after pupal recovery and the health of adult silkworms.

[0026] 2. This invention introduces the objective and statistically verifiable criterion of pupal body color change, along with a series of refined environmental control measures such as stepped temperature rise and fall, water replenishment, and periodic airflow circulation. This transforms the judgment of key physiological processes and environmental transition operations from subjective experience into standardized procedures, greatly improving the operability and repeatability of the method and ensuring the consistency of the treatment results.

[0027] 3. The dual-refrigeration strategy adopted in this invention reduces the reliance on single long-term extreme low temperature and maintains the physiological functions of the pupa through an intermediate recovery period. This not only reduces the risk of overall eclosion failure due to long-term irreversible damage, but its standardized operating procedures also facilitate large-scale application, indirectly improving the economy and applicability of the technology.

[0028] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0029] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0030] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0031] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0032] The specific statistical and calculation methods of this invention for the emergence rate, the duration of the peak emergence period, and the proportion of adult wing fully extended are as follows: The emergence rate was calculated based on the total number of pupae that had been treated in a batch of experiments. The number of adult insects that emerged was observed and recorded daily, and the final result was calculated as the percentage of adult insects to the total number of pupae.

[0033] The duration of the peak molting period is determined by counting the number of newly molted individuals in the population multiple times a day (e.g., morning, noon, afternoon, and evening) throughout the molting phase, plotting a curve showing the change in the number of molted individuals, and defining the continuous period in which the number of molted individuals remains above 80% of the total number of molted individuals as the peak molting period, and its duration is the peak period duration (unit: h).

[0034] The percentage of adults with fully extended wings was determined by visual inspection within 24 hours of emergence from all successfully emerged adults. Individuals with fully extended wings, smooth and uncurled wing edges, symmetrical wings, and wings at a normal angle to the body's longitudinal axis were considered to have fully extended wings. The percentage of these individuals out of the total number of successfully emerged adults in the same period was used to express the percentage.

[0035] All data were based on observations from at least three independent replicate trials, and their average was calculated to ensure the reliability and representativeness of the results.

[0036] Example 1 Healthy castor silkworm larvae were selected, and starting from the fifth instar, the rearing temperature was gradually decreased at a rate of 1°C per day, from an initial temperature of 26°C to 19°C, allowing the castor silkworms to successfully complete maturation and pupation in a low-temperature environment. Twelve hours after pupation, the pupae were placed in the first low-temperature environment for initial cryopreservation at 6°C for 65 days. During this period, environmental checks and ventilation were conducted every 15 days for 15 minutes, maintaining temperature fluctuations within ±1°C. After the first cryopreservation, the pupae were transferred to the first recovery environment for initial recovery culture, with an ambient temperature of 26°C, relative humidity controlled at 75%, airflow maintained at 0.2 m / s, and 11 hours of light at an intensity of 100 lux. Recovery culture continued until more than 80% of the pupae in the population changed from the light yellow of the early pupal stage to a uniform yellowish-brown, typically requiring about 12 hours. A stabilization transition was then implemented, with the ambient temperature decreasing from 26°C to 16°C at a rate of 0.8°C per hour and maintained at 16°C for 9 hours. The pupae were then transferred to a second low-temperature environment. The second low-temperature preservation was conducted at 6°C for 35 days, with environmental checks and ventilation performed every 15 days. After the second low-temperature preservation, the pupae were transferred to a final recovery environment for emergence culture, using a stepped heating method: first, they were placed at 19°C for 10 hours, then the temperature was increased to 23°C at a rate of 0.8°C per hour and maintained for 14 hours, and finally increased to 26°C at the same rate and maintained until emergence was complete. In the final recovery environment, starting from the 24th hour, a 12-hour light cycle was provided daily with a light intensity of 150 lux. During emergence culture, the pupae were placed in a single layer on the emergence frame, ensuring the distance between adjacent pupae was 1.8 times their maximum body diameter, and the emergence frame was rotated 180 degrees every 60 hours.

[0037] To demonstrate the effectiveness of this embodiment, the group using the method of Example 1 was compared with the group using the method of Comparative Example 1. The method of Comparative Example 1 was as follows: a traditional single long-term cold storage method was used: healthy castor silkworm larvae were selected and reared at a normal temperature range (25~28℃) until pupation. 12 hours after pupation, the pupae were placed directly in a low-temperature environment with a storage temperature of 6℃ for 100 days. During this period, environmental checks and ventilation were carried out every 15 days for 15 minutes, and the temperature fluctuation was maintained within ±1℃. After cold storage, the pupae were directly transferred to the final recovery environment at a temperature of 26℃, with 12 hours of light per day and a light intensity of 150 lux, until eclosion was completed.

[0038] The results showed that, after a cumulative storage period of 100 days, the emergence rate of the castor silkworm pupae in Example 1 reached 88%, the peak emergence period lasted for 60 hours, the proportion of adults with fully extended wings was 85%, and their activity level was normal. In contrast, after a cumulative storage period of 100 days, the emergence rate of the castor silkworm pupae in Comparative Example 1 was only 8%, the peak emergence period lasted for 110 hours, the proportion of adults with fully extended wings was 0%, and the adults exhibited low vitality and deformed emergence. This indicates that the combination of staged low-temperature treatment and recovery culture effectively balanced developmental inhibition and the maintenance of physiological activity, thereby improving the emergence success rate and adult quality.

[0039] Example 2 Based on Example 1, an additional water replenishment treatment was added during the first recovery culture process: when the pupae were cultured in the first recovery environment at 26°C for 6 hours, distilled water was atomized into fine droplets with a diameter of approximately 10 micrometers using a conventional ultrasonic nebulizer and evenly sprayed onto the surface of the pupae. At this time, the relative humidity in the microenvironment was temporarily increased and maintained at 92% for 12 minutes, after which the humidity was restored to the conventional 75% control. The remaining steps, including the pre-pupae cooling induction, the parameters and ventilation management for the two cryopreservations, the execution of the stabilization transition steps, the criteria for judging body color changes, the final recovery process of stepwise temperature increase, and the use and rotation operation of the eclosion rack, were all completely consistent with Example 1.

[0040] This embodiment incorporated a water replenishment treatment. After a cumulative storage period of 100 days, the emergence success rate of castor silkworm pupae further increased to 90%, the peak emergence period was shortened to 56 hours, the proportion of adults with fully extended wings increased to 88%, and the adults were generally more vigorous. This indicates that introducing active and controlled water replenishment treatment during the recovery period of double-layer cold storage can effectively alleviate physiological dehydration in pupae, significantly improving their subsequent cold storage tolerance and final emergence quality.

[0041] Example 3 Based on Example 2, within 1 hour after completing the water replenishment treatment and restoring normal humidity control, a periodic micro-airflow circulation was initiated, which was executed for two complete cycles. Each cycle lasted 60 minutes, specifically: for the first 50 minutes, the airflow speed was maintained at the normal flow rate of 0.2 m / s in the first recovery environment, and then for the next 10 minutes, the airflow speed was increased to 1.0 m / s; this periodic micro-airflow circulation was performed during the light exposure phase. All other steps and parameters, including pre-pupae cooling induction, two cryopreservation and ventilation management, water replenishment treatment during the first recovery culture, stabilization transition steps, body color judgment, the final recovery process of stepwise temperature increase, and the use and rotation operation of the eclosion frame, were completely consistent with Example 2.

[0042] By incorporating a controlled, periodic micro-airflow circulation scheme, the emergence success rate of castor silkworm pupae reached 90% after a cumulative storage period of 100 days. The peak emergence period was further shortened to 52 hours, and 90% of adult pupae achieved full wing extension. Furthermore, the emergence process was more concentrated and synchronized, and the adults exhibited high vitality. This demonstrates that introducing a specific pattern of periodic micro-airflow circulation after water replenishment effectively breaks the stagnant air boundary layer around the pupae, significantly improves gas exchange and environmental uniformity, creates a superior microenvironment for the physiological recovery of the pupae, and thus enhances the synchronicity, success rate, and quality of emergence.

[0043] Example 4 Based on Example 3, the execution period of the periodic micro-airflow circulation was adjusted differently: when the periodic micro-airflow circulation after water replenishment treatment needed to be executed during the dark phase of the first recovery environment, its airflow pattern was adjusted, that is, the high-velocity airflow (1.0 m / s) for 10 minutes in each cycle was replaced with a medium-velocity airflow with a duration extended to 20 minutes and a constant velocity of 0.45 m / s; while during the light phase, the periodic micro-airflow circulation was still executed according to the original scheme of Example 3, that is, maintaining the pattern of 0.2 m / s for the first 50 minutes and 1.0 m / s for the last 10 minutes of each cycle. All other steps and parameters, including the cooling induction before pupation, two low-temperature preservation and ventilation management, water replenishment treatment in the first recovery culture, stabilization transition steps, body color judgment, the final recovery process of stepwise heating, and the use and rotation operation of the eclosion rack, were completely consistent with Example 3.

[0044] By optimizing the airflow pattern during the dark phase, the emergence success rate of castor silkworm pupae reached 91% after a cumulative storage period of 100 days. The peak emergence period was shortened to 48 hours, and the proportion of adults with fully extended wings increased to 92%. Furthermore, the emergence process was highly concentrated, and the overall activity and coordination of the adults were better. This indicates that adjusting the airflow pattern from high intensity and short pulses to a lower intensity, longer duration, and stable medium flow rate during the dark resting period effectively reduces the interference of non-rhythmic physical disturbances on the recovery of the pupal physiological rhythms. While maintaining environmental uniformity, it better ensures the stability of the rhythmic operation of physiological metabolism (such as respiratory metabolism and hormone secretion), thereby further improving the synchronization, success rate, and health level of the adults during emergence.

[0045] Example 5 Based on Example 4, the eclosion culture method in the final recovery environment was specifically optimized: the pupae were placed in a single layer on a specially designed three-dimensional eclosion frame, which consisted of multiple layers of parallel plastic mesh plates with a vertical distance of 10 cm between each layer. The surface of the mesh plates was sandblasted to achieve a surface roughness Ra value of approximately 6.3 micrometers. When placing the pupae, the spacing between adjacent pupae was ensured to be 1.8 times their maximum body diameter. During the eclosion culture, the entire eclosion frame was rotated 180 degrees every 60 hours. All other steps and parameters, including pre-pupae cooling induction, two periods of cryopreservation and ventilation management, water replenishment and differentiated airflow circulation during the first recovery culture, stabilization transition steps, body color assessment, and the stepwise heating process, remained completely consistent with Example 4.

[0046] This optimized scheme for the physical layout and dynamic environmental exposure of pupae resulted in a 93% success rate in emergence of castor silkworm pupae after a cumulative storage period of 100 days. The peak emergence period was shortened to 48 hours, and 94% of adult pupae achieved full wing extension. The emergence process was highly synchronized, and the overall activity level of the population was consistent. This demonstrates that by placing the pupae in a single layer, maintaining spacing, using a three-dimensional support structure with a specific roughness, and periodically rotating the emergence frame, microenvironmental differences caused by fixed positions can be effectively eliminated, providing equal environmental stimulation to all pupae. This significantly improves the synchronicity, success rate, and uniformity of adult quality during emergence.

[0047] Example 6 Based on Example 5, the mesh plate of the emergence frame was further optimized: multiple shallow grooves matching the shape of the pupa were provided on the plastic mesh plate, each shallow groove being used to fix one pupa in place. The depth of the shallow grooves was controlled at about 40% of the average height of the pupa, and the inner surface of the shallow grooves was treated with the same sandblasting to maintain a roughness Ra value of about 6.3 micrometers. The shallow grooves were arranged in a neat array on the mesh plate, with the center distance between adjacent shallow grooves equal to 1.8 times the distance between adjacent pupae, and the opening size of the shallow grooves was slightly larger than the maximum diameter of the pupa to ensure that the pupa could be stably embedded in them without being completely enclosed. All other steps and parameters, including pre-pupae cooling induction, two low-temperature preservation and ventilation management, water replenishment and differentiated airflow circulation during the first recovery culture, stabilization transition steps, body color judgment, stepwise heating process, and periodic rotation operation during emergence culture, were completely consistent with Example 5.

[0048] By incorporating a shallow groove positioning structure, the emergence success rate of castor silkworm pupae reached 93% after a cumulative storage period of 100 days. The peak emergence period was further shortened to 45 hours, and 94% of adult pupae achieved full wing extension. Furthermore, no pupae were observed to slip or shift during multiple rotations of the emergence frame, demonstrating an extremely stable and synchronized emergence process, with all adults exhibiting high vitality. This indicates that the shallow groove structure, by providing precise local positioning and lateral support, significantly enhances the stability of the pupae during dynamic environmental adjustments, effectively preventing potential interference from positional shifts in the emergence process, thereby further improving the stability and ultimate success rate of the emergence process.

[0049] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A method for inhibiting the development of castor silkworm pupae using a double-layer cold storage system, characterized in that, Includes the following steps: S1: Starting from the fifth instar of the castor silkworm, gradually reduce the rearing temperature from 25~28℃ to below 20℃, so that the castor silkworm can complete the maturation and pupation in a low temperature environment; 12 hours after the castor silkworm pupates, place it in the first low temperature environment for the first low temperature preservation, the preservation temperature is 4~8℃, and the preservation time is 60~70 days. S2: After the first low-temperature preservation is completed, the castor silkworm pupae are transferred to the first recovery environment for the first recovery culture. The temperature of the first recovery environment is 24~28℃ and the recovery time is 12h until the pupae develop to the predetermined recovery stage. In the first recovery environment, the relative humidity is controlled at 70%~80%, and air circulation is maintained at an airflow speed of 0.1~0.3 m / s; during the first recovery culture, 10~12 hours of light is provided, with a light intensity of 50~150 lux. S3: After the first recovery culture is completed, the castor silkworm pupae are placed in a second low-temperature environment for a second low-temperature preservation at a temperature of 4~8℃ for 30~40 days. S4: After completing the second cryopreservation, the castor silkworm pupae are transferred to the final recovery environment for emergence culture until they complete emergence; The final recovery environment temperature is 25~28℃. Starting from the 24th hour of the final recovery environment, a 12-hour light cycle is provided every day, and the light intensity is controlled at 100~200 lux. During the low-temperature storage period, environmental checks and ventilation are carried out every 12 to 18 days in steps S1 and S3, with a ventilation time of 10 to 20 minutes. During this period, the temperature fluctuation of the low-temperature environment should not exceed ±1℃.

2. The method for inhibiting the development of castor silkworm pupae according to claim 1, characterized in that, In step S2, the criterion for determining when the pupae have developed to the predetermined recovery stage is that more than 80% of the pupae in the group change from the light yellow color of the early pupal stage to a uniform yellowish-brown color.

3. The method for inhibiting the development of castor silkworm pupae according to claim 1, characterized in that, During the first recovery culture process in step S2, after the pupa's body color reaches the predetermined recovery stage and before proceeding to step S3, a stabilization transition step is also included: the temperature of the first recovery environment is reduced from 24~28℃ to 15~18℃ at a rate of 0.5~1℃ / h, and maintained at 15~18℃ for 6~12h before being transferred to the second low-temperature environment.

4. The method for inhibiting the development of castor silkworm pupae according to claim 1, characterized in that, In the final recovery culture process of step S4, a step-by-step heating method is used: First, place the pupae that have just been taken out of the second low temperature environment at 18~20℃ for 8~12 hours; Subsequently, the temperature was increased to 20-25°C at a rate of 0.5-1°C / h and maintained for 12-16 hours; Finally, the temperature is increased to 25-28°C at a rate of 0.5-1°C / h and maintained until the eclosion is complete.

5. The method for inhibiting the development of castor silkworm pupae according to claim 1, characterized in that, During the first recovery culture in step S2, a water replenishment treatment is performed: water is atomized into droplets with a diameter of 5-15 micrometers using an atomizing device and sprayed evenly on the surface of the pupa. The relative humidity of the treatment environment is maintained at 90-95%, and each treatment lasts for 10-15 minutes. After treatment, the relative humidity is restored to the normal relative humidity of the first recovery environment of 70%-80%.

6. The method for inhibiting the development of castor silkworm pupae according to claim 5, characterized in that, Within 2 hours after completing the moisture replenishment treatment and restoring normal relative humidity control, a periodic micro-airflow circulation is initiated and executed, which is performed for 2 complete cycles. Each complete cycle lasts for 60 minutes, specifically: for the first 50 minutes, the airflow speed is maintained at the normal flow rate of the first restored environment, 0.1~0.3 m / s, and then for the next 10 minutes, the airflow speed is adjusted to a high-speed airflow of 0.8~1.2 m / s.

7. The method for inhibiting the development of castor silkworm pupae according to claim 6, characterized in that, During the first phase of environmental restoration under illumination, periodic micro-airflow circulation is performed; during the dark phase, after performing moisture replenishment and restoring normal relative humidity control, the airflow velocity of the periodic micro-airflow circulation is adjusted from 0.8~1.2m / s to 0.4~0.5m / s, and the duration is 20min.

8. The method for inhibiting the development of castor silkworm pupae according to claim 1, characterized in that, The specific implementation of transferring the castor silkworm pupae to the final recovery environment for eclosion culture in step S4 is as follows: the pupae are placed in a single layer on an eclosion frame with a three-dimensional support structure. The eclosion frame is composed of multiple parallel grid plates with a vertical distance of 8-12 cm between layers. The surface of the grid plates is made of an inert material with a roughness Ra value between 3.2 and 12.5 micrometers. When placing the pupae, ensure that there is a distance between adjacent pupae that is at least 1.5 to 2 times their own maximum body diameter. During the eclosion culture period, the eclosion frame is rotated 90 to 180 degrees every 48 to 72 hours.

9. The method for inhibiting the development of castor silkworm pupae according to claim 8, characterized in that, The molting frame has multiple shallow grooves on its grid plate that match the shape of the pupa. Each shallow groove is used to fix one pupa in place. The depth of the shallow groove is 30% to 50% of the average height of the pupa, and the inner surface roughness Ra value of the shallow groove is maintained in the range of 3.2 to 12.5 micrometers. The shallow grooves are arranged in an array on the grid plate. The center distance between adjacent shallow grooves is equal to 1.5 to 2 times the distance between adjacent pupae, and the opening size of the shallow groove is larger than the maximum body diameter of the pupa to ensure that the pupa is stably embedded without being completely enclosed.

10. The method for inhibiting the development of castor silkworm pupae according to claim 1, characterized in that, In step S1, the temperature is reduced from 25 to 28°C to below 20°C at a rate of 0.5 to 1.5°C / d.