Regulation and control method for synchronous eclosion of pupae in middle and later periods of silkworm cross variety

Abstract

The invention relates to a regulation and control method for synchronous eclosion of middle and later-stage pupae of a silkworm cross variety, and belongs to the technical field of silkworm improved variety breeding. The method aims at solving the problems that existing strong low-temperature inhibition causes pupa physiological injury and synchronous regulation and control in the middle and later periods are insufficient. According to the technical scheme, after cocoons are cut and pupae are identified in the middle stage of pupa body development, female and male pupa forms are observed every day, quantitative scoring is conducted, and comprehensive development scores of two groups are calculated and compared; therefore, differentiated temperature protection is performed on male pupa groups which develop quickly or slowly, and meanwhile, the residual development time difference of the two parties is estimated according to a pre-established development progress comparison table. When the time difference exceeds a set threshold value, physical stimulation is applied to the lagging party, and if the lagging party enters a key window before eclosion, a nutritional agent is synchronously sprayed and supplemented; and after the development of the two parties reaches the standard, differential photosensitive management is executed to trigger synchronous eclosion. The method is mainly used for improving eclosion synchronism and health degree of pupae in silkworm seed production, so that silkworm egg quality is improved.

Description

Technical Field

[0001] This invention belongs to the field of silkworm breeding technology, and more specifically, this invention relates to a method for regulating the synchronous emergence of pupae in the middle and late stages of crossbreeding of silkworms. Background Technology

[0002] In the field of silkworm breeding, the conventional method to achieve synchronous emergence of male and female moths of crossbred varieties is to implement multi-stage temperature control throughout the development process. Especially during the cocooning stage, individuals that develop faster are often subjected to low-temperature refrigeration at about 5°C to inhibit their development and forcibly delay their development process, thus achieving early preliminary time synchronization.

[0003] However, this strong cryogenic suppression method has a negative impact on pupal physiology. Even after the pupae are thawed and enter the critical mid-to-late stages of pupal development, the damage continues to manifest: male moths show scale and hair loss, decreased vitality, and a sharp reduction in mating ability and duration; female moths show restless oviposition and an increase in defective eggs, leading to a decrease in the effective egg circle rate and the rate of good eggs. The fundamental reason is that pupae are extremely sensitive to low-temperature stress, and the low-temperature damage suffered in the early stages is persistent, affecting subsequent normal development and the quality of emergence.

[0004] Therefore, in the mid-to-late pupal stage, which is crucial for determining the quality and synchronization of final emergence, there is an urgent need in this field for a method that can refine, rebalance, and physiologically repair the developmental progress of male and female pupae based on the aforementioned necessary early-stage regulation. Summary of the Invention

[0005] This invention addresses the aforementioned shortcomings by resolving the issues of physiological damage to pupae caused by strong cryoinhibition and insufficient synchronous regulation in the mid-to-late stages.

[0006] To achieve the above objectives, this invention provides a method for regulating the synchronous emergence of pupae in the middle and late stages of silkworm crossbreeding. After removing the cocoons and identifying the pupae, the following steps are performed daily at fixed time windows: S1. Morphological observation and quantitative evaluation: Observe the morphology of male and female pupae of the cross-breeding variety and quantitatively score them according to the preset coloring and hardness levels; S2. Development progress calculation and comparison: Calculate the comprehensive development score for the female pupa population and the male pupa population respectively. The comprehensive development score is obtained by averaging the scores of each quantitative scoring index in step S1, and then compare the two scores. S3. Synchronized Control Decision and Execution: Based on the comparison results of S2, the following two items are executed synchronously: (a) Differentiated temperature control: When the overall development scores of female and male pupa populations are different, the male pupa populations that develop faster or slower are protected by different environmental temperatures based on the comparison results; when the overall development scores of the two are the same, both female and male pupa populations are protected at a standard temperature of 25°C to 26°C. (b) Developmental lag judgment and intervention: Based on the comprehensive developmental score obtained from S1, query the pre-established developmental progress comparison table, estimate the remaining development days of the male and female pupal groups, and calculate the estimated remaining time difference ΔT between the two groups. If ΔT exceeds the set threshold, physical stimulation will be applied to the pupa population with delayed development within a fixed time window on that day. If, according to the development progress comparison table, the pupae with delayed development have entered the 48-72 hour period before emergence, then apply a nutrient supplement to them once within a fixed time window on that day. S4. Synchronized Emergence Management: When the overall development score of both male and female pupae is ≥10 points according to the development progress comparison table, and their morphology meets the requirements for photosensitive status, differentiated photosensitive management shall be implemented.

[0007] Preferably, the quantitative scoring in step S1 includes the following four indicators: A. Compound eye staining: brown is grade 1, black is grade 2, and jet black is grade 3; B. Antennae coloration: Yellow is level 1, black at first is level 2, and jet black is level 3; C. Color of the pupa's tail: yellow is grade 1, brown is grade 2, and dark brown is grade 3; D. Pupae hardness: Hard pupae are grade 1, soft pupae can be pressed down but do not dent are grade 2, and soft pupae can form a dent are grade 3.

[0008] Preferably, the differentiated temperature control in step S3(a) specifically includes: When the overall development score of the male pupa population is lower than that of the female pupa population, the male pupa population is protected at an ambient temperature of 27°C to accelerate its development. When the overall developmental score of the male pupa population is greater than that of the female pupa population, the male pupa population is protected at an ambient temperature of 24℃ to delay its development. The female pupa population is kept at a standard temperature of 25°C to 26°C.

[0009] Preferably, the developmental progress comparison table in step S3(b) is pre-established at a standard temperature of 25°C to 26°C; the developmental progress comparison table includes the correspondence between the comprehensive developmental score and the estimated remaining developmental days at the standard temperature.

[0010] Preferably, the threshold set in step S3(b) is 12 hours; the fixed time window is from 8:00 to 10:00 AM every day.

[0011] Preferably, the physical stimulation in step S3(b) is: on the day when ΔT exceeds the set threshold, apply a sound wave vibration stimulation with a vibration acceleration of 0.5 m / s² to 2.0 m / s² and a frequency of 30 Hz to 50 Hz to the pupal population with delayed development once, each time lasting 2 to 4 minutes.

[0012] Preferably, the sound wave vibration is applied by placing the vibration source 10 to 15 centimeters below the tray holding the silkworm pupa.

[0013] Preferably, the nutritional supplementation in step S3(b) is as follows: within the fixed time window, a glucose-containing aqueous solution is applied by spraying once; wherein the glucose mass percentage is 0.1% to 0.3%; the spray volume is 20 ml to 40 ml per square meter of the silkworm tray area holding the pupae; and the spraying is applied using a spraying device with an atomization particle size of 50 micrometers to 100 micrometers.

[0014] Preferably, the morphological characteristics of the photosensitive state described in step S4 are: the compound eyes and antennae are jet black, the tail is dark brown, and the pupa is soft and can form depressions.

[0015] Preferably, the differentiated photosensitive management in step S4 is as follows: on the day of emergence, the ambient temperature is maintained at 25°C to 26°C, and a light source is used to expose the female pupae to light 4-5 hours before mating and the male pupae to light 1 hour before mating. The light intensity is 50 lux to 200 lux, and the light source is a fluorescent lamp.

[0016] The present invention has at least the following beneficial effects: I. This invention employs a gentle temperature gradient regulation (adjusted only within the range of 24℃, 25-26℃, and 27℃) as the primary means of regulating developmental progress in the mid-to-late stages, thus avoiding irreversible damage such as cell membrane damage and metabolic disorders caused by extreme low temperatures. Through differentiated protection (e.g., using a slightly lower 24℃ to slow down the development of fast-developing male pupae and a slightly higher 27℃ to promote the development of slow-developing pupae), the metabolic rate can be gently regulated without exceeding the physiological tolerance range, achieving a "fine-tuning" of developmental progress. This fundamentally protects the physiological health of the pupae, laying the foundation for obtaining vigorous adults.

[0017] Second, by consulting a reference table, this invention can convert the score into an estimated remaining development time at a standard temperature. This process digitizes and standardizes the assessment of the developmental progress of both males and females and the estimation of their time difference (ΔT), providing a basis for subsequent regulatory decisions and reducing misjudgments caused by differences in human experience.

[0018] Third, based on quantitative diagnosis, this invention applies physical stimulation (vibration) with specific parameters to the lagging pupae when the time difference (ΔT) exceeds a set threshold (e.g., 12 hours). Simultaneously, if the lagging pupae have entered the critical pre-emergence window (e.g., 48-72 hours), nutritional supplementation (nebulized glucose) is applied concurrently. This may gently accelerate the development process without causing mechanical damage by promoting fluid circulation within the pupa and enhancing cellular metabolic activity. During the critical pre-emergence period, low-concentration glucose is supplemented via nebulization to provide a directly absorbable exogenous energy substrate for the energy-intensive process of pupal emergence. The combination of physical stimulation and nutritional supplementation aims to "boost" the lagging pupae from two dimensions: activating metabolism and providing energy, enabling them to safely and effectively catch up with their developmental progress in a short period.

[0019] 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. Attached Figure Description

[0020] Figure 1 This is a comparison chart of the mid-stage morphology, late-stage morphology, and morphology awaiting photosensitive exposure. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0022] 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.

[0023] The regulatory basis of this invention is the establishment of a "quantitative scoring system for pupa morphology" and a "developmental progress-time comparison table". This system grades and scores four key morphological indicators of silkworm pupae (compound eye coloring, antennae coloring, tail body color, and pupal body firmness), transforming subjective morphological observations into objective numbers, thereby better judging the developmental progress.

[0024] The "Development Progress-Time Comparison Table" was compiled by statistically analyzing observation data from over 10 batches of pupae, each containing at least 1000 pupae, of varieties such as 'Furong' and '932', under standard protective temperatures (25–26℃). The specific method involved observing and recording the morphological scores of large batches of pupae (typically ≥1000 pupae per batch) at a fixed time each day (e.g., 8:00 AM), calculating the average score for the population, and tracking their actual emergence dates to determine the average remaining development days at standard temperatures corresponding to different average score ranges. A typical correspondence in the comparison table is shown in Table 1 below.

[0025] Table 1: Based on the interval settings and statistical patterns in Table 1, the linear correspondence between each score interval is summarized in Table 2, which can be used to calculate the estimated number of remaining days for any score.

[0026] Table 2: The process of using this table to estimate the molting time difference (ΔT) and make decisions is as follows: Calculate the average score of the population: After daily observation, calculate the average developmental scores Pf and Pm of the female pupa population and the male pupa population in the crossbred varieties, respectively.

[0027] To determine the remaining development days: Based on the values ​​of Pf and Pm, refer to Table 1 (typical interval correspondence) and Table 2 (linear relationship explanation) to calculate the "estimated remaining development days" for both female and male pupae using linear interpolation, denoted as Df and Dm. For example, when Pf = 8.2 points (in the 7-9 point range), according to Table 2, for every 0.1 day increase in score within this range, the number of days decreases by 0.05 days. Starting from (7.0 points, 4.0 days), Df = 4.0 - (8.2-7.0) × 0.5 = 3.4 days (calculation basis: Pf = 8.2 points is in the 7.0-9.0 point range, and the linear relationship of 'for every 0.1 day increase in score, the number of days decreases by 0.05 days' applies to this range).

[0028] Calculate the estimated molting time difference: Estimated molting time difference ΔT = |Dm - Df| × 24 (unit: hours).

[0029] Judgment and Decision-Making: Set a synchronization threshold (e.g., 12 hours). If ΔT > 12 hours, it is determined that the developmental progress difference is significant, and physical stimulation or nutritional supplementation intervention should be initiated immediately for the developmentally lagging pupal population, depending on the specific circumstances. The trigger condition for physical stimulation is that ΔT exceeds the set threshold; the trigger condition for nutritional supplementation is that the developmentally lagging pupae have entered 48-72 hours before emergence. The two interventions can be implemented individually or in combination, depending on the conditions.

[0030] The basis for determining the 12-hour synchronization threshold (ΔT_threshold) is as follows: This threshold is a key intervention trigger point determined based on regression analysis of large-scale seed production data. Preliminary experiments and statistical analysis show that: 1. Quality Inflection Point Analysis: When the estimated eclosion time difference (ΔT) between male and female pupae is less than 12 hours, the vigor, mating ability, and egg quality of the male moths after eclosion remain at a high level with minimal fluctuations. When ΔT exceeds 12 hours, the ineffective waiting period of the first ecloding moth (especially the male moth) is significantly prolonged, leading to energy depletion, scale damage, and accelerated sperm aging. This results in a shortened mating duration and decreased fertilization efficiency, ultimately manifesting as a statistically significant decrease in the effective egg circle rate and the rate of good eggs (the decrease is usually greater than 10%).

[0031] 2. Trade-off between correction efficiency: When ΔT is within 12 hours, effective synchronization can be achieved within the following 2-3 days using only the differential temperature regulation (S3a) of this invention. If ΔT is greater than 12 hours, it indicates that the developmental differences are already significant, and physical stimulation and / or nutritional supplementation (S3b) must be initiated to achieve effective catch-up within the subsequent critical developmental window (approximately 2-4 days) and ensure synchronized molting.

[0032] 3. Variety verification: In the verification test of the cross between 'Furong×932' and '932×Furong', the 12-hour threshold can effectively distinguish between the two scenarios of "requiring mild adjustment" and "requiring strong intervention". After intervention, the synchronous emergence rate and seed production quality indicators of each combination have been steadily improved.

[0033] Therefore, setting the synchronization threshold to 12 hours is a comprehensive result that balances the necessity of regulation, the effectiveness of intervention, and the economics of operation, and is applicable to silkworm crossbreeding varieties.

[0034] Experimental basis for key intervention parameters: Physical stimulation parameters: Preliminary experiments showed that when the applied vibration acceleration was below 0.5 m / s², the promoting effect on pupal development was not significant; when the acceleration was above 2.0 m / s², some pupae exhibited decreased viability after emergence. Therefore, the preferred physical stimulation parameters were determined to be: vibration acceleration of 0.5-2.0 m / s², frequency of 30-50 Hz, and duration of 2-4 minutes. This parameter range can effectively promote the metabolism of pupae with developmental delays without causing mechanical damage or excessive stress.

[0035] Nutritional supplementation parameters: glucose solution concentration (0.1%-0.3%), spraying timing (48-72 hours before emergence), and atomization method (particle size 50-100 μm) have all been optimized through experiments to provide efficient and safe energy supplementation for pupae and avoid nutrient waste or excessively high environmental humidity.

[0036] Example 1 1. Test materials and pretreatment Crossbreeding varieties: Select the commonly used production varieties "Furong" (female) and "932" (male).

[0037] Source of seed cocoons: Selected from healthy and disease-free silkworm populations of the same age. On the 8th day after cocooning, the cocoons are cut and the sex is identified (pupa identification) by the pink color of the compound eyes.

[0038] Experimental setup: 1200 cocoons were randomly selected from a large batch of seed cocoons. After pupation, 500 female pupae and 550 male pupae were precisely selected and placed in a standard silkworm tray. The remaining pupae were discarded. The humidity of the experimental environment was controlled at 75%-85%.

[0039] 2. Detailed Explanation of Control Steps Day 1 (the day of pupal observation) 8:00 AM – 10:00 AM: S1. Morphological observation and quantitative assessment: Fifty pupae were randomly selected from both the female and male pupae groups as representative samples. Two experienced technicians independently observed and scored the pupae back-to-back, and the average value was taken to reduce subjective error.

[0040] Scoring criteria: A. Compound eye coloration: Grade 1 (brown), Grade 2 (black), Grade 3 (pitch black).

[0041] B. Antennae coloring: Level 1 (yellow), Level 2 (beginning to turn black), Level 3 (pitch black).

[0042] C. Tail body color of the pupa: Grade 1 (yellow), Grade 2 (brown), Grade 3 (dark brown).

[0043] D. Pupae hardness: Grade 1 (pupae are hard), Grade 2 (soft, can be pressed down but does not dent), Grade 3 (soft, can form a dent).

[0044] Example: If a male pupa has compound eyes of grade 2, antennae of grade 2, tail color of grade 2, and hardness of grade 2, then the score for a single pupa is 8 points.

[0045] Calculation results: The average score of the female pupa population, Pf, is 7.8 points; the average score of the male pupa population, Pm, is 8.3 points.

[0046] S2. Development progress calculation and comparison: Calculation shows that Pm>Pf, indicating that the current development progress of the male pupa population is slightly faster than that of the female pupa population.

[0047] S3. Synchronized Control Decision-Making and Execution: Based on the results of S2, the following two decisions and operations are executed synchronously: (a) Differential temperature control: Since male pupae develop faster (Pm>Pf), to slow down their development, the trays containing male pupae were moved to a constant temperature incubator at 24.0℃ for protection; the female pupae were placed in a constant temperature incubator at 25.5℃ (within the standard temperature range) for protection. All incubators were kept in complete darkness and had a relative humidity of 80%.

[0048] (b) Determination of developmental lag: Referencing the pre-established "Developmental Progress-Time Comparison Table" and calculating based on the linear relationship in Table 2: Pf = 7.8 points, falling between 7.0 points (4.0 days) and 8.0 points (3.5 days), Df = 4.0 - (7.8 - 7.0) × 0.5 = 3.6 days; Pm = 8.3 points, falling between 8.0 points (3.5 days) and 9.0 points (3.0 days), Dm = 3.5 - (8.3 - 8.0) × 0.5 = 3.35 days. Calculate the estimated molting time difference ΔT = |Dm - Df| × 24 = |3.35 - 3.6| × 24 = 6.0 hours.

[0049] Decision: Since ΔT = 6.0 hours < 12 hours (set threshold), physical stimulation and nutritional supplementation intervention will not be initiated this time.

[0050] 8:00 AM – 10:00 AM on the second day: S1. Morphological observation and quantitative assessment: repeated operation. Pf = 8.5 points, Pm = 8.9 points were measured.

[0051] S2 and S3. Synchronized control decision-making and execution: Based on the S2 results (Pm>Pf), we continued to implement (a) differentiated temperature control to maintain a protective environment of 24.0℃ for male pupae and 25.5℃ for female pupae.

[0052] Based on the S1 score, (b) developmental lag is assessed: Referring to the reference table and calculating: Pf = 8.5 points, Df = 3.5 - (8.5 - 8.0) × 0.5 = 3.25 days; Pm = 8.9 points, Dm = 3.5 - (8.9 - 8.0) × 0.5 = 3.05 days. Calculate ΔT = |3.05 - 3.25| × 24 = 4.8 hours.

[0053] Decision: If ΔT remains less than 12 hours, no intervention will be initiated.

[0054] Day 3, 8:00 AM – 10:00 AM: S1. Morphological observation and quantitative assessment: Repeated operation. Pf = 9.1 points, Pm = 9.4 points were measured.

[0055] S2 and S3. Synchronized control decision-making and execution: (a) Temperature control and (b) lag judgment are performed simultaneously. Referring to the reference table and calculating: Pf = 9.1 points, falling between 9.0 points (3.0 days) and 10.0 points (2.0 days), Df = 3.0 - (9.1 - 9.0) × 1.0 = 2.9 days; Pm = 9.4 points, Dm = 3.0 - (9.4 - 9.0) × 1.0 = 2.6 days. Calculate ΔT = |2.6 - 2.9| × 24 = 7.2 hours.

[0056] Decision: ΔT will decrease further; maintain temperature control and do not initiate intervention.

[0057] 8:00 AM – 10:00 AM on the 4th: S1. Morphological observation and quantitative assessment: Repeat operation. Scoring results: Pf = 10.2 points, Pm = 10.1 points. The morphology of all pupae met the "ready for light exposure" criteria: compound eyes and antennae were jet black, tail was dark brown, and the pupa was soft and concave.

[0058] S3. Synchronous Control Decision-Making and Execution: (b) Assessment of developmental lag: Refer to the reference table and calculate: Pf = 10.2 points, which falls between 10.0 points (2.0 days) and 12.0 points (1.0 day), Df = 2.0 - (10.2 - 10.0) × 0.5 = 1.9 days; Pm = 10.1 points, Dm = 2.0 - (10.1 - 10.0) × 0.5 = 1.95 days. Calculate ΔT = |1.95 - 1.9| × 24 = 1.2 hours.

[0059] Decision: Since ΔT is much smaller than the 12-hour threshold and both morphologies have met the criteria, no intervention will be initiated, and the process will directly switch to synchronous feathering management.

[0060] S4. Synchronous Feathering Management: All male and female pupae were placed in the same emergence chamber at 25.5℃.

[0061] Differential photosensitive treatment: On the scheduled mating day, fluorescent lights were turned on at 4:00 AM to expose the female pupae to light (the time interval between photosensitive treatment and mating was 5 hours); the male pupae were exposed to light at 8:00 AM (the time interval between photosensitive treatment and mating was 1 hour). The light intensity was controlled at 125 ± 10 lux.

[0062] 3. Effect Observation and Data Recording Emergence synchronicity: The measured difference in emergence time ΔT between male and female moths was 4.9 hours, indicating good synchronicity. This is because the female pupa can be controlled to emerge 3-5 hours before the male pupa, thus achieving a good emergence synchronicity effect.

[0063] Moth body weight: Male moths have intact scales and are active; female moths have outstretched wings and full abdomens.

[0064] Mating situation: Mating took place at 9:00 AM. Pairing was quick and stable, with no instances of couples breaking apart within 4 hours.

[0065] Egg-laying quality: After the pairs were separated, each moth laid eggs. The effective egg circle rate was 83.3%, and the average good egg rate was 94.5%.

[0066] Example 2 1. The test materials and pretreatment were the same as in Example 1.

[0067] 2. Detailed Explanation of Control Steps (Focusing on Key Differences) Preliminary Operations (Days 1-2): Same as Example 1, perform routine monitoring and decision-making for S1, S2 and S3.

[0068] After observation and calculation on the 3rd day, it was found that due to the initial difference, ΔT reached 16.8 hours (>12-hour threshold), and the female pupae were the ones with delayed development (i.e., Pm>Pf), and the female pupae with delayed development had entered the 48-72 hour window before emergence.

[0069] Day 3, 8:00 AM – 10:00 AM: S1. Morphological observation and quantitative assessment: Pf = 8.0 points, Pm = 9.2 points were measured.

[0070] S2 and S3. Synchronized control decision-making and execution: (a) Differential temperature control: Since male pupae develop faster (Pm>Pf), to slow down their development, the trays containing male pupae were moved to a constant temperature incubator at 24.0℃ for protection; the female pupae were placed in a constant temperature incubator at 25.5℃ (within the standard temperature range) for protection. All incubators were kept in complete darkness and had a relative humidity of 80%.

[0071] (b) Assessment and intervention for developmental delay: Consult the reference table and calculate: Pf = 8.0 points, falling within the 7.0-9.0 point range, Df = 3.5 days; Pm = 9.2 points, falling within the 9.0-10.0 point range, Dm = 3.0 - (9.2-9.0) × 1.0 = 2.8 days. Calculate ΔT = |2.8 - 3.5| × 24 = 16.8 hours > 12 hours.

[0072] Decision: Since ΔT exceeds the set threshold, and according to the comparison table, the female pupae with delayed development (Df=3.5 days, about 84 hours) have not entered the 48-72 hour window before emergence, only physical stimulation is initiated; after another 24 hours, it will be the second day (84-24=60 hours) before entering the 48-72 hour window before emergence, and then nutritional supplementation intervention will be carried out.

[0073] Physical stimulation: A vibration platform was used, with a vibration acceleration of 0.5 m / s² and a frequency of 30 Hz. The tray containing the female pupae was placed on the vibration platform and vibrated continuously for 2 minutes. The distance between the vibration source and the bottom of the tray was 10 cm.

[0074] Nutritional supplementation: Prepare a 0.1% glucose aqueous solution. Use an ultrasonic sprayer with a particle size of 50 micrometers to spray the female pupae, controlling the spray volume to 20 ml per square meter of the silkworm tray containing the pupae.

[0075] Day 4 and beyond: Continue daily observation. On Day 4, ΔT was measured to have decreased to 9 hours. On Day 5, the female pupa population score surpassed that of the others, and both reached the photosensitive state (score ≥ 10 points).

[0076] S4. Synchronous feathering management: During photosensitive management, the light intensity is controlled at 50 lux, and other aspects are the same as in Example 1.

[0077] 3. Effect Observation The feathering time difference ΔT was successfully corrected to 4.5 hours, with good synchronization.

[0078] The effective oviposition rate was 82.6%, and the average rate of good oviposition eggs was 95.4%.

[0079] It is evident that the method of this invention can effectively correct developmental deviations and achieve seed production quality superior to that of traditional methods.

[0080] Example 3 1. The test materials and pretreatment were the same as in Example 1.

[0081] 2. Detailed Explanation of Control Steps (Focusing on Key Differences) On the second day, it was found that the male pupae were significantly delayed in development, with ΔT reaching 18 hours.

[0082] 8:00 AM – 10:00 AM on the second day: S1. Morphological observation and quantitative assessment: Pf = 9.0 points, Pm = 7.5 points were measured.

[0083] S2 and S3. Synchronized control decision-making and execution: (a) Differential temperature regulation: Since Pf>Pm (male pupae are lagging), the male pupae population was moved to 27.0℃ (the upper limit of accelerated development).

[0084] (b) Assessment and intervention for developmental delay: Consult the reference table and calculate: Pf = 9.0 points, Df = 3.00 days; Pm = 7.5 points, Dm = 4.0 - (7.5 - 7.0) × 0.5 = 3.75 days. Calculate ΔT = |3.75 - 3.00| × 24 = 18 hours > 12 hours.

[0085] Decision: Since ΔT exceeds the set threshold, physical stimulation intervention will be initiated immediately for the male pupae with delayed development. At the same time, according to the control table, it is determined that the male pupae (Dm=3.75 days, about 90 hours) have not entered the 48-72 hour window before emergence. After another 24 hours, they will enter the 48-72 hour window before emergence on the second day (90-24=66 hours), and then nutritional supplementation intervention will be carried out.

[0086] Physical stimulation: Vibration acceleration was set to 2.0 m / s², frequency to 50 Hz, duration to 4 minutes, and distance of vibration source to 15 cm.

[0087] Nutritional supplementation: Prepare a glucose aqueous solution with a concentration of 0.3%, a spray volume of 40 mL / m², and an atomized particle size of 100 micrometers.

[0088] Day 3 and onwards: Daily observation continued. The male pupa's development accelerated significantly after the intervention, and the ΔT was measured to have decreased to 10 hours on the 3rd. On the 4th, both pupae simultaneously entered the photosensitive state.

[0089] S4. Synchronous feathering management: When exposed to light, the light intensity is 200 lux.

[0090] 3. Effect Observation The feathering time difference ΔT was successfully corrected to 3.8 hours, with good synchronization.

[0091] The effective oviposition rate was 84.9%, and the average rate of good oviposition eggs was 97.8%.

[0092] The pupae are viable after emergence.

[0093] Comparative Example 1 It does not perform the quantitative scoring and mild adjustment as described in this invention. When male pupae are found to be growing faster after pupal identification, they are directly placed in a cold storage at 5.0 ± 0.5℃ for 48 hours, and then taken out and restored to 25.5℃ for protection.

[0094] Result: Male moths lost a large number of scales and hairs, their wings curled slightly, and they moved sluggishly.

[0095] Male moths are less vigorous during mating, and the average mating time is only 2.8 hours, with early separation of pairs.

[0096] The female moth is impatient when laying eggs, and the eggs are scattered.

[0097] The effective ovipositor rate dropped to 48.4%, and the average rate of good ovipositors was only 60.2%.

[0098] It is evident that extreme low temperatures cause irreversible physiological damage, severely impairing seed production quality.

[0099] Comparative Example 2 It performs steps S1, S2, and S3(a) of the present invention (morphological scoring and temperature regulation), but when S3(b) determines that ΔT > 12 hours, physical stimulation and nutritional supplementation intervention are not performed. Steps S1, S2, S3(a), and S4 are the same as in Example 2.

[0100] After observation and calculation on the 3rd day, Pf = 7.9 points and Pm = 9.3 points were measured (i.e., Pm > Pf, indicating female pupae are lagging). Referring to the reference table, Df = 3.55 days and Dm = 2.7 days were obtained. The calculated ΔT = |2.7 - 3.55| × 24 = 20.4 hours > 12 hours, but only temperature control was implemented.

[0101] The delayed pupae not only have a slow correction speed when only temperature is fine-tuned, but also have poor vitality of male and female moths during mating, even though the synchronization of emergence is improved by S4 photosensitive management.

[0102] The final feathering time difference ΔT was controlled within the range of 5-7 hours, which was not concentrated enough.

[0103] Male moths that emerge too early already show some decline in vitality.

[0104] The effective oviposition rate was 70.5%, and the average rate of good oviposition eggs was 84.9%.

[0105] It is evident that while mild temperature regulation has some effect, the male moths exhibit poor vitality, which affects the quality of mating.

[0106] Comparative Example 3 Experienced technicians visually assess the rate of development and implement differential temperature protection. There is no standardized scoring system, no scores are calculated, and no reference table is used to look up ΔT.

[0107] result: Different technicians made vastly different judgments, resulting in varying timing and intensity of adjustments.

[0108] The effective ovarian circle rate averaged only 65.2%, and the rate of good eggs fluctuated greatly.

[0109] Comparative Example 4 The procedure is the same as in Example 2, but when performing the physical stimulation of S3(b), the vibration acceleration is increased to 3.0 m / s², the frequency is 60 Hz, and the duration is 5 minutes.

[0110] Results: Approximately 15% of the stimulated pupae showed lethargy after emergence, and the proportion of short-winged moths increased by 5%.

[0111] Although it did not affect synchronization, the quality of the moths decreased. The rate of good eggs dropped to 83.7%.

[0112] Comparative Example 5 The procedure is the same as in Example 2, but the nutrient supplementation in S3(b) is changed to be carried out 20 hours before molting, and direct drip irrigation is used instead of atomized spraying.

[0113] Result: The timing of replenishment was too late, and energy preparation was insufficient.

[0114] Drip irrigation can cause localized over-wetting, leading to the suffocation or mold growth of a small number of pupae.

[0115] The effective ovarian circle rate and the rate of good ovarian cells were both lower than those in Example 2.

[0116] The comparison data between the above embodiments and comparative examples are shown in Table 3: The present invention provides a method for regulating the synchronous emergence of silkworm pupae in the middle and late stages of mating. Through strategies such as morphological quantitative assessment, differential temperature fine-tuning, gentle physical stimulation and precise nutritional supplementation, it effectively overcomes the physiological damage caused by low-temperature refrigeration in the prior art, achieves the synchronization of emergence that meets mating requirements, and can also improve mating quality.

[0117] In the embodiments and comparative examples of this invention, the "effective ovarian circle rate" and "average good ovarian circle rate" were all measured using the following uniform method: 1. Mating and spawning management: After emerging as moths, they mate at 9:00 AM.

[0118] The mating environment is maintained at a temperature of 25–26℃, a relative humidity of 75%–85%, and dim lighting.

[0119] After mating lasted for 4 hours, the pairs were manually separated at 1:00 p.m.

[0120] 2. Single moth oviposition and egg ring collection: After separating the pairs, each female moth is placed individually in an oviposition circle to lay eggs.

[0121] The spawning environment is completely dark, with a temperature of 25–26°C and a humidity of 75%–85%, and the spawning period is about 18–24 hours.

[0122] After the female moth has finished laying eggs, it is removed to obtain a framed seed of a single moth egg ring.

[0123] 3. Criteria for determining effective ovarian follicles: Effective egg zone: An egg zone with ≥400 eggs laid by a single moth, and where the eggs are relatively concentrated and there are no obvious missing eggs.

[0124] Ineffective ovipositors: ovipositors with less than 400 eggs, or ovipositors with extremely scattered or severely clustered eggs.

[0125] Formula for calculating effective egg zone rate: Effective egg zone rate (%) = (Number of effective egg zones / Total number of egg-laying female moths) × 100%.

[0126] 4. Criteria and statistics for determining good eggs: Five sample areas (28 circles per area) were randomly selected from each valid ovipositor.

[0127] Good eggs: The eggs are plump, the shells are smooth, the color is uniform (the inherent color of the variety), and the contents are plentiful.

[0128] Poor quality eggs include hatching eggs (hatched 7-10 days after hatching), unfertilized eggs (not changing color after hatching), dead eggs (abnormal color, sunken), and deformed eggs.

[0129] Formula for calculating average good egg rate: ; Where n is the number of effective ovipositors.

[0130] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for regulating the synchronous emergence of pupae in the middle and late stages of silkworm crossbreeding, characterized in that, After identifying the pupae by removing the cocoon during the mid-stage of pupal development, the following steps should be performed daily at fixed times: S1. Morphological observation and quantitative evaluation: Observe the morphology of male and female pupae of the cross-breeding variety and quantitatively score them according to the preset coloring and hardness levels; S2. Development progress calculation and comparison: Calculate the comprehensive development score for the female pupa population and the male pupa population respectively. The comprehensive development score is obtained by averaging the scores of each quantitative scoring index in step S1, and then compare the two scores. S3. Synchronized Control Decision and Execution: Based on the comparison results of S2, the following two items are executed synchronously: (a) Differentiated temperature control: When the overall development scores of female and male pupa populations are different, the male pupa populations that develop faster or slower are protected by different environmental temperatures based on the comparison results; when the overall development scores of the two are the same, both female and male pupa populations are protected at a standard temperature of 25°C to 26°C. (b) Developmental lag judgment and intervention: Based on the comprehensive developmental score obtained from S1, query the pre-established developmental progress comparison table, estimate the remaining development days of the male and female pupal groups, and calculate the estimated remaining time difference ΔT between the two groups. If ΔT exceeds the set threshold, physical stimulation will be applied to the pupa population with delayed development within a fixed time window on that day. If, according to the development progress comparison table, the pupae with delayed development have entered the 48-72 hour period before emergence, then apply a nutrient supplement to them once within a fixed time window on that day. S4. Synchronized Emergence Management: When the overall development score of both male and female pupae is ≥10 points according to the development progress comparison table, and their morphology meets the requirements for photosensitive status, differentiated photosensitive management shall be implemented.

2. The method according to claim 1, characterized in that, The quantitative scoring in step S1 includes the following four indicators: A. Compound eye staining: brown is grade 1, black is grade 2, and jet black is grade 3; B. Antennae coloration: Yellow is level 1, black at first is level 2, and jet black is level 3; C. Color of the pupa's tail: yellow is grade 1, brown is grade 2, and dark brown is grade 3; D. Pupae hardness: Hard pupae are grade 1, soft pupae can be pressed down but do not dent are grade 2, and soft pupae can form a dent are grade 3.

3. The method according to claim 1, characterized in that, The differentiated temperature control described in step S3(a) specifically includes: When the overall development score of the male pupa population is lower than that of the female pupa population, the male pupa population is protected at an ambient temperature of 27°C to accelerate its development. When the overall developmental score of the male pupa population is greater than that of the female pupa population, the male pupa population is protected at an ambient temperature of 24℃ to delay its development. The female pupa population is kept at a standard temperature of 25°C to 26°C.

4. The method according to claim 1, characterized in that, The developmental progress comparison table mentioned in step S3(b) is pre-established at a standard temperature of 25°C to 26°C; the developmental progress comparison table includes the correspondence between the comprehensive developmental score and the estimated remaining developmental days at the standard temperature.

5. The method according to claim 1, characterized in that, The threshold set in step S3(b) is 12 hours; the fixed time window is from 8:00 AM to 10:00 AM every day.

6. The method according to claim 1, characterized in that, The physical stimulation described in step S3(b) is as follows: on the day when ΔT exceeds the set threshold, apply a sound wave vibration stimulation with a vibration acceleration of 0.5 m / s² to 2.0 m / s² and a frequency of 30 Hz to 50 Hz to the pupal population with delayed development once, each time lasting 2 to 4 minutes.

7. The method according to claim 6, characterized in that, The sound wave vibration is applied by placing the vibration source 10 to 15 centimeters below the tray holding the silkworm pupa.

8. The method according to claim 1, characterized in that, The nutritional supplementation in step S3(b) is as follows: within the fixed time window, a glucose-containing aqueous solution is applied by spraying once; wherein the glucose mass percentage is 0.1% to 0.3%; the spray volume is 20 ml to 40 ml per square meter of the silkworm tray area holding the pupae; and the spraying is applied using a spraying device with an atomization particle size of 50 micrometers to 100 micrometers.

9. The method according to claim 1, characterized in that, The morphological characteristics of the photosensitive state described in step S4 are: the compound eyes and antennae are jet black, the tail is dark brown, and the pupa is soft and can form depressions.

10. The method according to claim 1, characterized in that, The differentiated photosensitive management in step S4 is as follows: on the day of emergence, the ambient temperature is maintained at 25°C to 26°C, and a light source is used to expose the female pupae to light 4-5 hours before mating and the male pupae to light 1 hour before mating. The light intensity is 50 lux to 200 lux, and the light source is a fluorescent lamp.

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