A method for artificially inducing diapause of the mulberry pyralid
By raising mulberry borers in transparent plastic boxes, controlling temperature and replacing mulberry leaves, screening and preserving diapause mulberry borers, the problem of diapause mulberry borers in the laboratory was solved, and the effective diapause effect of mulberry borers was achieved, supporting the prevention and control of mulberry borers and the research on diapause mechanism.
Patent Information
- Application Number
- CN202310037777.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-01-10
- Publication Date
- 2025-08-05
- Estimated Expiration
- 2043-01-10
AI Technical Summary
The existing technology is difficult to efficiently induce mulberry diapause in the laboratory, which leads to difficulty in collecting experimental materials, affecting mulberry borer prevention and control and diapause mechanism research.
By feeding mulberry borers of 3-4 years of age in a transparent plastic box, the temperature is controlled at 16℃, the feces and old mulberry leaves are cleaned, fresh mulberry leaves are replaced every 3 days, transparent plastic paper is added on the 10th day, and the silk-spiking mulberry borers are observed at 27℃ after 21 days. After 7 days, mulberry borers that have entered diapause state are screened and placed at 8-16℃ for storage.
The diapause rate of mulberry borer has reached 80%, which is easy to operate, low cost, stable and feasible, and is suitable for studying the mechanism of mulberry borer borer.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure SMS_3
Abstract
Description
Technical Field
[0001] The invention relates to an artificial breeding technology for insects, in particular to a method for artificially inducing diapause of mulberry borer. Background Art
[0002] Mulberry trees are a vital component of the sericulture industry. They contain a variety of beneficial ingredients for humans and animals, and can be used as traditional Chinese medicine and feed additives. However, mulberry trees are constantly threatened by pests and diseases, which severely harm their normal growth and development. The mulberry borer is one of the most serious mulberry pests worldwide.
[0003] The mulberry borer is a Lepidoptera insect with a complete metamorphosis. Its larvae have five instars. Instars 1-3 are more harmful to the tender leaves and new buds of mulberry trees. Instars 4-5 have a huge appetite and excrete rapidly, causing serious damage to mulberry leaves. They can also excrete feces containing various pathogens, which can contaminate the leaves. The mulberry borer has good environmental resistance, making it ubiquitous in mulberry gardens worldwide. In the cold season, the mulberry borer can overwinter by diapausing as mature larvae, and then continue metamorphosis and pupation into moths in the spring, achieving the continuation of the population. Therefore, the overwintering diapause of the mulberry borer is a very important factor affecting the normal growth of mulberry leaves in the following year. Current research on the overwintering diapause of the mulberry borer is relatively limited. The difficulty lies in that there is only a fixed period each year to find wild diapause mulberry borers, making it impossible to effectively collect experimental materials. Therefore, we hope to prepare materials for this special period by artificially inducing diapause of mulberry borer indoors. In this way, we can provide the required experimental samples in large quantities simply, quickly and easily, and provide guarantees for subsequent prevention and control of mulberry borer and research on the molecular biological mechanisms behind diapause. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a method for artificially inducing diapause of mulberry borer in a laboratory with simple operation, low cost and stable technology in view of the above-mentioned situation.
[0005] To solve the above problems, the present invention is a method for artificially inducing diapause of mulberry borer, which comprises the following steps:
[0006] (1) Prepare a transparent plastic box of appropriate size according to the feeding amount and punch a small hole in the lid.
[0007] (2) Take an appropriate amount of wild 3-4-year-old mulberry borers and place them together with mulberry leaves in a plastic box.
[0008] (3) Place the mulberry borer and the plastic box in a constant temperature incubator at 16°C with no humidity or light-dark ratio requirements. Clean the mulberry borer feces and old mulberry leaves every 3 days and add fresh mulberry leaves.
[0009] (4) On the 10th day, place suitable transparent plastic paper in the plastic box and place it in the order of "one mulberry leaf - mulberry borer - one plastic paper". Continue to clean up the old mulberry leaves and mulberry borer feces every 3 days.
[0010] (5) On the 21st to 30th day, observe from the side of the plastic paper and place the mulberry borer that spins silk, has a shortened body, and refuses to feed on mulberry leaves in a 27°C incubator.
[0011] (6) Place the mulberry borer in an incubator at 27°C for 7 days. If the borer does not show signs of molting and pupation, it is considered to have entered the diapause state. The borer that has entered the pupation state must be discarded.
[0012] (7) Placing the diapause mulberry borer at a temperature of 8-16℃ can preserve the diapause mulberry borer for a long time.
[0013] Preferably,
[0014] In step (2), the age is 4 years old.
[0015] In step (5), the number of days is 21 days.
[0016] In step (3), there are no requirements for the humidity and light-dark ratio of the breeding environment.
[0017] In addition, placing the diapause-inducing mulberry borer at a temperature of 8-16°C can preserve the diapause-inducing mulberry borer for a long time.
[0018] Experiments have shown that the method of the present invention can induce diapause in mulberry borers, achieving a successful diapause rate of up to 80%. This method can induce diapause in mulberry borers by simply regulating the rearing temperature. Furthermore, the diapause effect can be verified by returning the temperature to normal, eliminating short-term dormant borers and avoiding experimental errors. The method of the present invention is simple to operate, highly feasible, has a low technical threshold, and provides stable results, making it suitable for researchers studying the diapause mechanism of mulberry borers. DETAILED DESCRIPTION
[0019] Example 1
[0020] (1) Prepare a transparent plastic box of appropriate size according to the feeding amount and punch a small hole in the lid.
[0021] (2) Take an appropriate amount of wild 4th-instar mulberry borers and place them together with fresh mulberry leaves free of insect bites and diseases in a plastic box.
[0022] (3) Place the mulberry borer and the plastic box in a constant temperature incubator at 16°C with no humidity or light-dark ratio requirements. Clean the mulberry borer feces and old mulberry leaves every 3 days and add fresh mulberry leaves.
[0023] (4) On the 10th day, place suitable transparent plastic paper in the plastic box and place it in the order of "one mulberry leaf - mulberry borer - one plastic paper". Continue to clean up the old mulberry leaves and mulberry borer feces every 3 days.
[0024] (5) On the 21st day, observe from the side of the plastic paper and place the mulberry borer that has spun silk, shortened its body, and refused to feed on mulberry leaves in a 27°C incubator.
[0025] (6) Place the mulberry borer in an incubator at 27°C for 7 days. If the borer does not show signs of molting and pupation, it is considered to have entered the diapause state. The borer that has entered the pupation state must be discarded.
[0026] The above steps were repeated three times, and the statistical diapause rate was as shown in Table 1:
[0027] Table 1
[0028]
[0029] Example 2
[0030] According to step (2), 5 groups of 1st, 2nd, 3rd, 4th and 5th age moths were set, with 30 moths in each group. The other conditions were the same as those in Example 1. The moths were reared, and the diapause rates at different age stages were shown in Table 2.
[0031] Table 2
[0032]
[0033]
[0034] It can be seen from this that 1-2 instar mulberry borers have poor low temperature tolerance and slow growth rate, making it difficult to successfully induce diapause in mulberry borers at the right time; 5-instar mulberry borers have entered the final stage of development, and their metabolites and genes cannot quickly adapt to low temperature stimulation and enter stable diapause conditions. Most 5-instar mulberry borers re-pupate in a short recovery temperature of 27°C, that is, the diapause state is unstable; 3-4 instars are more suitable for low temperature-induced diapause, can effectively enter a stable diapause state and basically do not affect growth and development.
[0035] Example 3
[0036] According to step (3), the temperatures were set at 1°C, 4°C, 8°C, 12°C, 16°C, 18°C, 20°C, and 27°C, with a total of 8 groups, 30 mulberry borers in each group. The other conditions were the same as in Example 1. The diapause rates are shown in Table 3.
[0037] Table 3
[0038] temperature Deaths pupation number diapause number Diapause rate 1℃ 30 0 0 0.0％ 4℃ 30 0 0 0.0％ 8℃ 22 0 8 26.7％ 12℃ 6 0 24 80.0％ 16℃ 1 2 27 90.0％ 18℃ 0 4 26 86.7％ 20℃ 0 10 20 66.7％ 27℃ 0 30 0 0.0％
[0039] This indicates that the diapause rate of 4th-instar mulberry borers can reach over 80% at 12-18°C. Temperatures too low can easily cause death, while higher temperatures can cause the borers to pupate and fail to enter a stable diapause state. However, it should be noted that at 12°C, the growth and development of mulberry borers is slower than at 16°C, and experimental results show that this can also cause greater mortality. Therefore, considering the diapause rate and time cost, 16°C is the optimal diapause-inducing temperature for 4th-instar mulberry borers.
[0040] Example 4
[0041] According to step (3), the relative humidity of the incubator environment was set to 20%, 50% and 90%, totaling 3 groups, with 30 mulberry borers in each group. The other conditions were the same as in Example 1. The diapause rates are shown in Table 4.
[0042] Table 4
[0043] relative humidity Deaths pupation number diapause number Diapause rate 20％ 2 1 27 90.0％ 50％ 3 3 24 80.0％ 90％ 2 3 25 83.3％
[0044] It can be seen from this that the relative humidity of the environment has no obvious effect on the diapause rate of mulberry borer and is not the main factor inducing diapause of mulberry borer.
[0045] Example 5
[0046] According to step (3), the light-dark ratio (L:D / h) of the incubator lighting time was set to 24:0, 12:12, and 0:24 for a total of 3 groups, with 30 mulberry borers in each group. The other conditions were the same as in Example 1. The diapause rates are shown in Table 5.
[0047] Table 5
[0048] Light-dark ratio / h Deaths pupation number diapause number Diapause rate 24:0 1 2 27 90.0％ 12:12 1 3 26 86.7％ 0:24 2 1 27 90.0％
[0049] It can be seen from this that light duration has no obvious effect on the diapause rate of mulberry borer and is not the main factor inducing diapause of mulberry borer.
[0050] Example 6
[0051] According to step (5), the low temperature treatment was set to 7 days, 14 days, 21 days and 30 days, and the mulberry borers that spun silk, had shortened bodies and refused to feed on mulberry leaves were removed. There were 30 mulberry borers in each group. The other conditions were the same as in Example 1. The diapause rates are shown in Table 6.
[0052] Table 6
[0053]
[0054] It can be seen from this that after 21 days, the diapause rate is basically stable. In order to save time and cost, 21 days is appropriate.
[0055] Example 7
[0056] According to step (6), the temperature was set to 27°C for 3 days and then returned to 16°C, the temperature was returned to 16°C after 7 days of treatment, and the temperature was returned to 16°C after 14 days of treatment. There were 3 groups in total, with 30 mulberry borers in each group. The number of mulberry borers with diapause characteristics before 27°C treatment, the number of mulberry borers that continued to diapause on the last day of treatment, and the number of mulberry borers that continued to diapause within 10 days after returning to 16°C were counted. The other conditions were the same as in Example 1. The data are shown in Table 7.
[0057] Table 7
[0058]
[0059] Therefore, a 14-day treatment at 27°C would cause the mulberry borer to exit diapause, which is too long. A 3-day treatment might result in some delayed development that may not be detected immediately, and they may continue to pupate when returned to 16°C. Selecting diapause-inducing mulberry borers treated for 3 days would affect experimental accuracy to a certain extent. Therefore, a 7-day treatment at 27°C is more effective for screening mulberry borers that have entered stable diapause.
[0060] Example 8
[0061] According to step (7), the cryopreservation temperature was set at 1°C, 4°C, 8°C, 16°C, and 20°C. Thirty stable diapause mulberry borers were selected from each group and placed in plastic boxes. After one month, the number of deaths (placed at room temperature for 20 minutes, the head and tail of the mulberry borer were gently stimulated with a sharp object, and no reaction was considered death) and the number of diapause were counted, as shown in Table 8.
[0062] Table 8
[0063]
[0064] It can be seen from this that 1℃ and 4℃ can easily cause death of mulberry borer, and long-term storage at 20℃ will release diapause and induce pupation. Therefore, the temperature for long-term storage of mulberry borer in diapause state should be 8-16℃.
Claims
1. A method for artificially inducing diapause of mulberry borer, comprising the following steps: (1) Prepare a transparent plastic box according to the feeding amount and punch a small hole in the lid; (2) Take wild mulberry borers at the 3rd or 4th instar stage and place them together with mulberry leaves in a plastic box; (3) Place the mulberry borer and the plastic box in a constant temperature incubator at 16°C, with no humidity or light-dark ratio requirements. Clean the mulberry borer feces and old mulberry leaves every 3 days and add fresh mulberry leaves. (4) On the 10th day, place transparent plastic paper in the plastic box and place it in the order of "one mulberry leaf - mulberry borer - one plastic paper". Continue to clean up the old mulberry leaves and mulberry borer feces every 3 days. (5) On the 21st to 30th day, observe from the side of the plastic paper and place the mulberry borer that has spun silk, shortened its body, and refused to feed on mulberry leaves in an incubator at 27°C; (6) Place the moth in a 27°C incubator for 7 days. If the moth does not show signs of molting and pupation, it is considered to be in diapause. The moth should be discarded if it has entered the pupation state. The moth can be kept in diapause for a long time by placing it at a temperature of 8-16°C.
2. The method according to claim 1, wherein: In step (2), the age is 4 years old.
3. The method according to claim 1, wherein: In step (5), the number of days is 21 days.