CN114521537B - Lugworm diapause induction method and long-term storage method - Google Patents
Lugworm diapause induction method and long-term storage method Download PDFInfo
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Abstract
The invention relates to an lugworm diapause induction method and a long-term storage method. The lugworm diapause induction method comprises the following steps: (1) Selecting Armoracia chinensis in a sensitive insect state as an object for diapause induction regulation; (2) The lugworm selected in the step 1) is placed in a diapause inducing environment, and the lugworm is induced to enter a diapause state by regulating and controlling the combination of the illumination time, the temperature period and the total inducing time per day. During long-term storage, lugworm subjected to diapause induction is stored under original induction conditions. The method can induce more than 85% of arma bug adults to enter diapause by selecting a proper sensitive insect state, regulating and controlling the illumination time length, the temperature period and the total induction time length every day, has the advantages of simple operation method, easy mastering and use, low cost, high efficiency and convenient popularization and application, realizes economical and efficient long-term storage of arma bugs, obviously reduces the large-scale feeding time and labor cost of the arma bugs, and is favorable for industrial large-scale production and efficient release and utilization of the arma bugs.
Description
Technical Field
The invention belongs to the technical field of biological control of agricultural pests, and particularly relates to an arma chinensis diapause induction method and a long-term storage method.
Background
The arma chinensis is an excellent predatory natural enemy insect which is widely distributed in China and has the characteristics of wide predation range, high reproductive capacity, strong adaptability and the like. Can be used for catching more than 40 kinds of common pests with multiple diseases in agriculture and forestry of Lepidoptera, coleoptera, hymenoptera, hemiptera and the like. At present, researches on morphology, biology, artificial feed, predation capacity, propagation and utilization of the arma bug are carried out at home and abroad, and the arma bug also has a remarkable prevention and control effect in application of pests such as tobacco, vegetables, fields, gardens and the like. The method for breeding large amount of colony of Armoracia chinensis by indoor use of Armoracia chinensis instead of prey such as tussah pupa or noctuid larva is simple, low in economic cost, and convenient for large-scale propagation, and is a natural enemy product with great commercial production and application potential. When the arma bug is actually used as a natural enemy product, the same problems as other natural enemy insect products are faced, such as long production period, short product storage period, inconsistent product and prevention and control requirements, and the like, so that the delivery period of the natural enemy insect product is often difficult to be consistent with the pest emergence period, no natural enemy is available when pests occur greatly, and the like, and industrial production and large-scale application of the arma bug are severely restricted.
At present, the storage time of natural enemy insects is prolonged by mainly adopting a refrigeration technology so as to ensure that the insects are still in an optimal thrown state before being released by individuals. However, the cold resistance of different insect species has certain difference, the refrigeration method is not suitable for all natural enemy products, and is influenced by the development state of insects, and the refrigeration time is limited. In addition, the quality of the refrigerated natural enemy product is often reduced, the survival or reproductive ability is reduced, the prevention and control efficiency is greatly reduced, and the prevention and control effect of the product is reduced. Therefore, the method for effectively prolonging the shelf life of the natural enemies is developed by utilizing the development characteristics of the natural enemies to improve the self stress resistance of the natural enemy insects while ensuring the reasonable external conditions of large-scale propagation and application of the natural enemies, and has important significance for solving the problems.
In the field of entomology research, some insects can enter a diapause state, the development rate of the insects is obviously reduced, the cold resistance is obviously improved, the insects are inactive or have few activities, the diapause state can be generally maintained for weeks or even more than one year, and the insects can still grow and develop after the diapause is relieved. There are many factors affecting diapause, and various factors affect each other, and for different insects, the factors affecting diapause are often different, so how to select the diapause condition of a specific insect is a technical difficulty. However, whether adult arma chinensis winters in a diapause state or not, which are main environmental factors influencing diapause, and which developmental stage of arma chinensis is most sensitive to environmental stimulation are not researched and reported at home and abroad at present. In the research on the aspect, it is generally considered that the determination of the lugworm diapause induction environmental condition parameters and the determination of diapause states are very difficult.
Disclosure of Invention
In view of the problems of the prior art, the present invention provides a diapause induction method and a long-term storage method of lugworm. More than 85% of adult arma chinensis can be induced to enter diapause by finely regulating and controlling diapause inducing conditions of arma chinensis, the arma chinensis can be stored for more than 150 days, the operation method is simple, and the method has the advantages of easiness in mastering and using, low cost, high efficiency, convenience in popularization and application and the like, economical, efficient and long-term storage of arma chinensis is realized, the large-scale feeding time and labor cost of arma chinensis are remarkably reduced, and the method has important guiding significance for industrial large-scale production and efficient release and utilization of arma chinensis.
The technical scheme for solving the technical problems is as follows:
an lugworm diapause induction method comprises the following steps:
(1) Selecting Armoracia chinensis in a sensitive insect state as a diapause induction regulation object;
(2) Placing the arma chinensis selected in the step 1) in a diapause inducing environment, and inducing the arma chinensis to enter a diapause state by regulating and controlling the combination of the illumination time, the temperature period and the total inducing time each day.
The invention has the beneficial effects that:
the lugworm stinkbug diapause process can be regulated and controlled indoors through regulation of the sensitive insect state, temperature, photoperiod and duration, long-term storage of lugworm stinkbug is achieved, the technical bottleneck restricting large-scale feeding and propagation expanding application of lugworm stinkbug is solved, and the effect of the lugworm stink bug in green prevention and control of pests is improved.
The arma bug diapause induction method provided by the invention can induce more than 85% of arma bug adults to enter diapause, prolongs the preservation time, has the advantages of simple operation method, easiness in mastering and use, low cost, high efficiency, convenience in popularization and application and the like, realizes economic, efficient and long-term storage of arma bugs, obviously reduces the time and labor cost for large-scale breeding of arma bugs, and has important guiding significance for industrial large-scale production and efficient release and utilization of arma bugs.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, in the step (2), the illumination time per day adopts the following conditions: the light treatment is carried out for 8-10 hours in the daytime and the dark treatment is carried out for 16-14 hours at night.
The beneficial effect who adopts above-mentioned scheme is: the adoption of the conditions can induce the adult arma chinensis to enter a diapause state, delay the development rate of the adult arma chinensis, delay the spawning time and greatly prolong the storage period and the product service life of the adult arma chinensis.
Further, in the step (2), the conditions of the warm period include: the light period is 15 ℃, and the dark period is 5 ℃.
The beneficial effect who adopts above-mentioned scheme is: the inventor unexpectedly finds that setting different temperatures in the light period and the dark period, and setting the temperature in the light period to be higher than that in the dark period is beneficial to improving the diapause rate.
Further, in the step (2), the total induction time is more than 30 days. Preferably, the total induction period is more than 40 days.
The beneficial effect who adopts above-mentioned scheme is: the inventor unexpectedly discovers in research that the diapause rate of the lugworm bugs can reach 100% in the total induction time of more than 30 days, and the death rate of the lugworm bugs after diapause release can be remarkably reduced in the total induction time of more than 40 days.
Further, the relative humidity of the diapause inducing environment is 75 +/-5%, and the illumination intensity is 66%.
The beneficial effect who adopts above-mentioned scheme is: the adoption of the humidity condition is favorable for improving the survival rate of the arma chinensis, and the adoption of the illumination intensity condition is favorable for reducing energy consumption and saving cost.
Further, in the step (1), the sensitive insect state of lugworm diapause induction regulation is initial eclosion imago, and lugworm in the insect state is selected as a diapause induction object. Preferably, arma chinensis of adults within 2 days of primary emergence is selected as the subject of diapause induction regulation.
The beneficial effect who adopts above-mentioned scheme is: the inventor unexpectedly discovers in research that different development stages have different sensibility and induction rate to diapause of lugworm bugs, eggs and nymphs of 1-5 years cannot develop to the next bug state and die finally under the diapause induction condition, adults on different days of eclosion have different results on induction stimulation, and unexpectedly discovers that the lugworm bugs of which the adults are initially eclosion (particularly the lugworm bugs of which the adults within 2 days of initial eclosion) are most sensitive to environmental induction stimulation and are in the sensitive period of diapause induction, and only under the short illumination condition, the adults at the stage can enter the diapause, and the diapause rate can reach 85-90%.
Preferably, newly emerged adults (within 12 hours) are most sensitive to environmental induction stimuli.
The invention provides a long-term storage method of arma chinensis, which comprises the following steps: continuously placing the arma chinensis subjected to diapause induction in a diapause induction condition for storage, for example: and storing the mixture in a photoperiod L =8, a temperature period of 15 ℃/5 ℃, relative humidity of 75 +/-5% and illumination intensity of 66%. Or further storing the Armoracia chinensis subjected to diapause induction at 5-10 deg.C.
The beneficial effect who adopts above-mentioned scheme is: the storage conditions can delay the development rate of the imagoes, delay the spawning time and greatly prolong the storage period and the service life of the imagoes.
Further, the storage time is 0-220 days. Preferably, the storage time is from 0 to 150 days, most preferably, the storage time is from 90 to 150 days.
The beneficial effect who adopts above-mentioned scheme is: within the storage time range, the survival rate of the arma chinensis is higher, and the reproduction and predation capacity of the arma chinensis is still higher after the storage is finished; if the storage time is too long, the death rate of the arma chinensis is increased, and the spawning capacity is reduced.
Drawings
FIG. 1 is the comparison result of ovary and ovary tubule of Armoracia chinensis diapause imago and normal development imago in example 1 of the present invention; in fig. 1, a is an ovarian micrograph of lugworm female adults diapause; b is an ovarian tubule microscopic picture of lugworm female imagoes in diapause; c is an ovary microscopic picture of normal developmental lugworm female imagoes; d is the ovarian tubule microscopic picture of normal development arma chinensis female imagoes.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
In order to solve the technical bottleneck of long-term storage in large-scale production of the arma bug, the invention provides a method for inducing the arma bug to enter a diapause state, which defines the sensitive insect state induced by diapause of the arma bug, masters the diapause regulation and control technology and realizes long-term storage of the arma bug.
An lugworm diapause inducing method and a long-term storage method comprise the following steps:
(1) The lugworm diapause type is reproductive diapause, adults within 2 days (within 0-48 hours) of initial eclosion are a sensitive period for diapause induction, and lugworm bugs in the period are selected as subjects for inducing diapause.
(2) The environmental conditions for inducing the lugworm to enter diapause are as follows: the temperature cycle T/C =15 ℃/5 ℃ (illumination period 15 ℃, dark period 5 ℃); photoperiod 8-10L (8-10 hours of light), 16-14D (14-16 hours of darkness); the induction time is longer than 30 days.
(3) Under the condition, lugworm diapause rate is about 80-90%.
(4) Keeping the lugworm in diapause state under diapause inducing condition for long-term storage.
The determination of the environmental condition parameters of the diapause induction of the arma bug and the judgment of the diapause state are very difficult, the inventor tests the influence of the combination conditions of constant temperature and different temperature on the diapause induction of the arma bug in different development age periods under different photoperiods through a large number of repeated experiments, samples adults under different induction conditions every 5 days to dissect and observe the ovarian development progress, compares the ovarian development progress with adults under normal development conditions, finally, unexpectedly screens out the environmental condition parameter combination with higher diapause induction rate, and determines the induction days for the adults to enter the diapause.
Arma chinensis is a predatory natural enemy insect, and in general, adults can survive for about 30 days under the condition of normal-temperature propagation expansion after self-emergence. The inventor unexpectedly finds in the research that the initial eclosion imagoes are most sensitive to the diapause induction stimulation, the period is very short and difficult to capture, and the time point is the best period for diapause induction, and the period is missed and the initial eclosion imagoes cannot be induced to enter the diapause. The induction diapause is carried out on the eclosion adults at the same time, which is beneficial to ensuring that the diapause adults have a consistent storage period, and the development uniformity is ensured after the storage is finished, thus being beneficial to the release and application of the diapause adults. The activity and the food intake of the arma chinensis in the diapause state are obviously reduced compared with those of normal adult development, so that the food and manual feeding cost is greatly saved.
Aiming at lugworm bug which is an excellent predatory natural enemy insect, the technical research of regulating and controlling the development progress of lugworm bug by using factors such as photoperiod, thermocycles and the like is developed, and the precise regulation and control of the lugworm bug development progress are realized through the systemic research of diapause induction and storage technology. The arma chinensis is stored in a diapause state, and the shelf life of the products is prolonged to more than 150 days from the original 30 days. By utilizing diapause regulation and control technology, the service life of the arma bug can be effectively prolonged, the reproduction expanding capability and efficient release utilization of the arma bug can be improved, the industrial production of natural enemy products such as the arma bug is promoted, stable product supply is provided for the biological control of pests in China, and the green prevention and control level of agricultural pests in China is improved.
The technical solution of the present invention is further illustrated by the following specific examples.
In the examples, the insect sources to be tested and the breeding method were as follows:
the arma bug population is a long-term breeding population in a laboratory where the inventor is located, and can be acquired by public for a non-commercial purpose to duplicate the test method described in the embodiment of the invention.
The raising conditions of eggs, nymphs and adults are as follows: the temperature is 26 +/-1 ℃, the relative humidity is 75 +/-5%, and the photoperiod is 1698D, and the feed is fed by tussah pupa.
Example 1 determination of Armoracia chinensis diapause induction sensitive period
Arma chinensis is an incomplete metamorphosis predatory natural enemy insect, the development process comprises eggs, nymphs (5 years old altogether), and adults, and the inventor guesses that the nymphs at 5 years old before the adults or the adult stage may be most sensitive to environmental induction stimuli. In order to determine the sensitivity of different insect states of the lugworm bugs to diapause induction, determine the optimal treatment time of diapause induction, and respectively perform diapause induction treatment on nymphs of the lugworm bugs in different insect states and different instars, the specific test method is as follows.
Performing induction treatment on Armoracia chinensis in different development stages under different photoperiods (S1 =8L, S2= 10L.
The test results are shown in table 1, and the results show that under the diapause induction condition, eggs and nymphs of 1 to 5 years can not develop to the next worm state and finally die. But after diapause induction, the adult insects have the phenomena of growth retardation and long-time non-oviposition. Further performing induction stimulation on adults emerging for different days and time points, and finding that the newly emerged adults (within 12 hours) are most sensitive to environmental induction stimulation, and the adults can enter diapause only under the condition of short illumination in a matching way, wherein the diapause rate can reach 85-90%. As shown in figure 1, after adult imagoes are sampled and subjected to a large amount of physiological dissection every 5 days, the ovaries of lugworm diapause female are not developed (A), and no follicles exist in small ovarian tubes (B), but lugworm bugs with the same development duration normally develop the ovaries of female worms and are mature (C), and a large amount of mature follicles exist in small ovarian tubes (D). As shown in FIG. 1, the ovary of lustinus chinensis female imago in diapause is in an immature state, no yolk deposition occurs, and the small ovary tube is in a transparent state; while the ovary of the normally developed female adult develops and matures, and a large number of mature ova are arranged in the small ovary tube.
TABLE 1 sensitivity and diapause induction rate of lugworm bug at different developmental stages (15 ℃/5 ℃ C.)
| Insect status | Egg | Age 1 | Age 2 | Age 3 | Age 4 | Age 5 | Imago |
| S1 | 0 | 0 | 0 | 0 | 0 | 0 | 90％±0.27 |
| S2 | 0 | 0 | 0 | 0 | 0 | 0 | 85％±0.35 |
Note: s1 refers to photoperiod 8l; s2 means 10l.
Example 2 diapause induction and Long-term storage of Armadma chinensis
In the embodiment, the diapause induction rate of the lugworm bugs under different conditions is determined by adjusting and controlling the combination of the photoperiod (namely the illumination time length every day), the temperature period and the induction treatment time length, and the environmental condition parameters with the highest induced diapause rate are screened out. The result shows that Armoracia chinensis initial emergence imagoes can induce Armoracia chinensis about 90% of the Armoracia chinensis population to enter diapause state under the temperature and light combination condition of 15 deg.C/5 deg.C, photoperiod L8: D16, and inducing duration of 40 days (total inducing time). The lugworm in diapause state can be stored for 90-150 days under the original inducing condition. The details of the test are as follows.
Example 2.1 screening of optimal conditions for diapause induction
2.1.1 diapause inducing Effect of photoperiod and thermocycle
Setting three-level repeatability tests, and carrying out the repeatability tests in a climatic chamber (Ningbo south RZX500C type), wherein the photoperiods are respectively 8L; the temperature cycle is 10 ℃/5 ℃,15 ℃/5 ℃, and the constant temperature is 18 ℃ and 26 ℃; relative humidity is 75 +/-5%; the illumination intensity is 66%. Adult males and females after primary emergence (within 12 hours) are paired and then are raised in a transparent plastic cup (250 ml) with a folded paper sheet, and the mouth of the cup is covered with gauze to prevent escape of the test insects. Each treatment was repeated 5 times, 10 pairs of adults were repeated, and 50 pairs of adults were treated in total (100 heads). The feeds were observed and changed day by day until the end of the test.
Test results of the effect of combined conditions of thermocycle and photoperiod on lugworm diapause induction:
the diapause rate of adult arma chinensis under different temperature and light cycle combination conditions is shown in table 2, and the adult arma chinensis cannot enter diapause at high temperature of 18 ℃ and 26 ℃ regardless of short illumination or long illumination. However, under the condition of day and night temperature change (namely higher temperature in the day and lower temperature at night), the armadilla chinensis can be induced to enter diapause, the illumination duration is shortened along with the day, and the diapause rate is obviously increased. Compared with two temperature-changing conditions, the excessively low temperature can be found to be unfavorable for survival and diapause of the adult arma bug, under the condition that the temperature cycle is 10 ℃/5 ℃, the illumination is carried out for 8 hours, the diapause rate is only 12 percent, the illumination is carried out for 10 hours, the diapause rate is increased to 40 percent, and is increased by 3.3 times.
Comprehensively, under the condition of a temperature cycle of 15 ℃/5 ℃, the diapause rate of the adult arma bug is the highest, 85% of individuals can be induced to enter diapause by short illumination for 10 hours, and when the illumination is shortened to 8 hours, the diapause rate can be improved to 90%. In practical application, armoracia chinensis imagoes can be treated under the condition of 15 deg.C/5 deg.C and illumination time of 8-10 h to induce Armoracia chinensis imagoes to enter diapause.
TABLE 2 diapause induction rate of Armoracia chinensis under different temperature cycle and photoperiod combination conditions
Note: data in the table are mean. + -. Standard error
The inventor unexpectedly discovers in research that under the condition of consistent other conditions, the temperature cycle is adjusted to be 15 ℃/15 ℃, and experimental results show that the death rate of the lugworm bugs is high at 15 ℃, and adults can not be induced to enter diapause at 15 ℃. The study unexpectedly discovers that the regulation of the thermocycles according to the different photoperiods has an important influence on the induced diapause result of the lugworm.
2.1.2 Effect of duration of diapause Induction treatment
On the basis of the test results, screening out appropriate conditions with higher diapause induction rate, namely 15/5 ℃, L8: D16 and relative humidity of 75 +/-5%; the illumination intensity is 66%, lugworm is placed under the condition of screening for continuous induction treatment for 10 days, 20 days, 30 days and 40 days respectively. And observing and recording the early spawning stage and the number of spawning individuals of the female insects until the test is finished. Each treatment 12 pairs of adults.
Test results of the effect of the length of diapause induction treatment:
on the basis of the above test, the appropriate conditions for diapause induction were selected as 15/5 ℃ and L8: D16, and the influence of different induction periods (10 days, 20 days, 30 days, and 40 days) on the rate of diapause induction was measured under these conditions. The results show (table 3) that after continuous treatment for 30 days under diapause inducing conditions, the diapause rate of lugworm bugs can reach 100%. After diapause individuals are transferred to normal development conditions (photoperiod L =16, temperature 26 ℃) to release diapause, survival conditions after diapause release are observed, and the death rate of adult ludinus chinensis in the diapause induction 30-day treatment group after diapause release is about 41.7%, but the death rate in the diapause induction 40-day treatment group is only 8.3%, so that the more suitable diapause induction treatment time is more than 40 days. The factors of diapause induction effect and economic cost are comprehensively considered, and the better diapause induction condition parameters in production and application are 15 ℃/5 ℃ of temperature cycle, L8: D16 of light cycle and more than 40 days of continuous induction time. Under the condition, adult arma chinensis entering diapause is induced and continuously stored under the inducing condition, and the adult arma chinensis diapause can be stored for more than 150 days. Normal adult Armoracia chinensis can be stored at low temperature of 15 deg.C for 30 days, and the storage life of Armoracia chinensis in diapause state is increased by more than 4 times.
TABLE 3 influence of duration of induction on Armoracia chinensis diapause induction rate
By combining the test results, the invention determines that the diapause induction sensitive treatment period of the lugworm is initial eclosion imago, and at the moment, diapause induction stimulation is carried out on the lugworm, so that the lugworm can be successfully induced to enter the diapause state. By carrying out different treatment combinations on three factors of photoperiod, temperature period and induction duration and repeated tests, diapause induction condition parameters with higher diapause induction rate are screened out: the temperature cycle is 15 ℃/5 ℃, the photoperiod L (8-10) is D (16-14), the induction time is longer than 40 days, and the diapause rate of the adult armadilla chinensis can reach 85-90% under the induction condition.
The invention provides a method for storing arma chinensis for a long time, which comprises the following steps: armoracia chinensis adults are induced under diapause inducing conditions (15 ℃/5 ℃, photoperiod L8: D16) for 40 days to enter diapause, and then are stored under the conditions for more than 150 days.
After the stored adult arma chinensis is transferred to normal development conditions (26 ℃, photoperiod L16: D8) to relieve diapause, the survival rate can reach more than 80 percent, and normal predation and spawning reproduction can be realized.
The invention also provides a long-term storage method of arma chinensis, which comprises the following steps: the adult lugworm induced into diapause under diapause inducing condition (15 deg.C/5 deg.C, light cycle L8: D16) is stored at low temperature of 5-10 deg.C for at least 200 days.
Arma bug normal development imagoes can be stored for only 30 days at low temperature of 15 ℃, and the diapause regulation technology of the invention improves the storage period of arma bug imagoes products from the original 30 days to more than 150 days, even more than 200 days, for example: the inventor prolongs the storage period of the adult arma chinensis products from the original storage period (stored at low temperature of 15 ℃) to 140.11 +/-6.37 days, the longest storage period can reach about 220 days, and the promotion effect is obvious. By utilizing the diapause induction and storage technology, the lugworm bugs can be stored for a long time, the propagation reserve amount of products is increased, and sufficient natural enemy products are timely provided to meet the prevention and treatment requirements when pests occur.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. An lugworm diapause induction method is characterized by comprising the following steps:
(1) Selecting Armoracia chinensis in a sensitive insect state as a diapause induction regulation object; the sensitive insect state of lugworm stinkbug diapause induction regulation is initial emergence imago, and lugworm stinkbug in the insect state is selected as diapause induction object;
(2) Placing the arma chinensis selected in the step (1) in a diapause induction environment, and inducing the arma chinensis to enter a diapause state by regulating and controlling the combination of the illumination time, the temperature period and the total induction time per day; the illumination time per day adopts the following conditions: the light treatment is carried out for 8-10 hours in the daytime and the dark treatment is carried out for 16-14 hours at night; the conditions of the warm cycle include: the light period is 15 ℃, and the dark period is 5 ℃; the total induction time is more than 30 days.
2. The lugworm bug diapause induction method of claim 1, wherein in step (2), the total induction time is more than 40 days.
3. The lugworm bug diapause induction method of claim 1, wherein the diapause induction environment has a relative humidity of 75 ± 5% and an illumination intensity of 66%.
4. The lugworm diapause induction method of any one of claims 1 to 3, wherein adult lugworm within Argus armatus in 2 days of initial emergence is selected as a subject for diapause induction regulation.
5. The method for storing the arma chinensis for a long time is characterized by comprising the following steps: continuously storing the lugworm bug induced by diapause in any one of claims 1-4 under the induction condition or at the temperature of 5-10 ℃.
6. The method of claim 5, wherein the storage period is 0 to 220 days.
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