A method for gradient low temperature acclimation and constant low temperature storage of adult acrosternum hilare

Abstract

The application discloses a kind of gradient low temperature domestication, constant low temperature storage method of acrosternum hilare adult, comprising the following steps: (1) 10-15 day old acrosternum hilare adult is placed into storage box, the substrate and armyworm are loaded in the storage box;(2) the storage box is placed into artificial climate chamber and gradient low temperature domestication is carried out, and the gradient low temperature domestication program is: 26 DEG C →20 DEG C domestication 8h →15 DEG C domestication 8h →10 DEG C domestication 8h →6 DEG C domestication 8h →4 DEG C domestication 8h;(3) after domestication, acrosternum hilare adult is placed at 4 DEG C and refrigerated, and refrigeration time is 40-60 days.The application can guarantee normal fecundity and egg hatch rate, and better offspring development and offspring survival rate in its life period by placing acrosternum hilare adult into storage box loaded with substrate and armyworm and carrying out gradient low temperature domestication, and then placing it at 4 DEG C and refrigerating for 40-60 days.

Description

Technical Field

[0001] This invention relates to a method for the gradient low-temperature domestication and constant low-temperature storage of adult stink bugs, belonging to the field of stink bug storage technology. Background Technology

[0002] The white-bellied bug (Arma chinensis Fallou), belonging to the order Hemiptera, family Pentatomidae, subfamily Armatainae, and genus Armata, is an excellent natural enemy insect in agriculture and forestry, characterized by its wide predation range and strong adaptability. This bug is distributed in Beijing, Tianjin, Gansu, Guizhou, Hebei, Heilongjiang, Hubei, Hunan, Jiangsu, Jiangxi, Jilin, and Yunnan provinces of my country. It preys on more than 40 common and prevalent agricultural and forestry pests, including those in the orders Lepidoptera, Coleoptera, Hymenoptera, and Hemiptera. As a highly valuable predatory natural enemy, domestic and international scholars have primarily studied its morphology, biology, predation characteristics, artificial rearing techniques, and preliminary field pest control effects. Since successfully introducing and raising black bugs in 2015, the Guizhou Provincial Tobacco Company has established 10 propagation sites in Suiyang, Fenggang, Zhenyuan, Anlong, Xingren, Dejiang, Xixiu, Qianxi, and Shuicheng counties within the province. They have acquired mature black bug propagation techniques, achieved large-scale breeding, and piloted the technology in 17 tobacco-producing areas across Guizhou and nationwide. Black bugs have played a significant role in the green control of pests in tobacco fields, achieving remarkable control effects. Therefore, the development and utilization of black bug products has excellent prospects. In the practical application of predatory insects such as black bugs, the storage of these predatory products is often a crucial factor determining the successful application of these insects. Before being released in large quantities, natural enemy insects must accumulate a sufficient amount of product within a certain period of time to meet application needs. However, the storage time of most natural enemy insects is generally short, and the propagation of indoor natural enemy populations is limited by the release time in the field and the target pests. As a result, the production period of natural enemy products is often difficult to coincide with the occurrence period of pests, leading to the death of natural enemies before the outbreak of pests, or the absence of natural enemies when pests are rampant.

[0003] Currently, refrigeration technology is mainly used to extend the storage time of natural enemy insects. Gao Zhuo (2010) studied the storage of black bugs. Adult black bugs were collected from the wild in spring and autumn, placed in a rearing box containing about 12 cm of damp sand, covered with leaves, and refrigerated at a low temperature of 4-6℃ for 4-5 months. The survival rate was about 90%. In winter, they can also be stored outdoors. A pit about 20-30 cm deep is dug in a sunny place, filled with 15-20 cm of damp fine sand, and the black bugs are placed in it. After covering with fallen leaves, the pit is covered with a mesh screen. The survival rate of the black bugs is still about 90% after 3-5 months of storage. For revival, a gradient heating method is used. The black bugs are first placed at 15℃ for 1-2 days, and then placed in a rearing room with a temperature of 23-28℃ and a relative humidity of 70%. However, this storage method is for wild-collected stink bug populations that have overwintered or have not yet finished overwintering, and the operation is relatively complicated. When storing artificially propagated stink bug products, they cannot be used directly, and when the quantity of products is large, the operation takes a long time and is inefficient.

[0004] Patent document (CN109757441A) discloses a method for storing bugs, which includes: 1) cleaning and disinfecting the storage site: preparing an artificial climate chamber for insect rearing, cleaning and disinfecting the artificial climate chamber, and then letting it air dry for later use; 2) preparing the storage environment: setting one to six isolation net frames in the artificial climate chamber of step 1), the isolation net frames being horizontally arranged, and the intervals between two or more isolation net frames being the same; 3) gradient cooling treatment: placing bugs into the artificial climate chamber of step 2). 4) Low-temperature storage of the bugs: Set the artificial climate chamber in step 3) to a temperature of 5℃~20℃, humidity of 35%~75%RH, and light ratio of 16:8~14:10 (L:D) and store the bugs under these conditions; 5) Providing food for the bugs and cleaning the storage area: Inspect and replace the bug rearing cups in step 4) every 5~7 days; clean the artificial climate chamber every 25~30 days. This method can achieve bug storage for 60 days with a survival rate of over 50%.

[0005] Patent document (CN111316981A) discloses a method for cold-preserving adult stink bugs. The method includes: (1) selecting adult stink bugs that have emerged 20 to 100 hours ago in a rearing room; (2) placing the adult stink bugs selected in step (1) in a cold storage at 10°C to 15°C for 48 to 96 hours; (3) placing the adult stink bugs from step (2) in a storage box and storing them in a cold storage at 3°C ​​to 7°C, with damp cotton placed on top of the storage box to maintain moisture; (4) removing the storage box from step (3) and placing it in a cold storage at 10°C to 15°C for 2 to 6 hours, then placing it in the rearing room for feeding for 12 to 24 hours. This method can preserve stink bugs for up to 90 days with a survival rate of over 70%.

[0006] The above solutions primarily focus on how to preserve the black bug to ensure its survival rate. However, while the survival rate of black bugs under cold storage is important for black bug control, ensuring normal oviposition and hatching rates throughout their lifespan is even more crucial. Normal oviposition and hatching rates guarantee further propagation and achieve better control effects. However, the existing technologies mentioned above do not address this issue. Summary of the Invention

[0007] Based on the above, the present invention provides a method for the gradient low-temperature domestication and constant low-temperature storage of adult stink bugs, which can ensure the oviposition capacity and egg hatching rate of adult stink bugs, thereby overcoming the shortcomings of the prior art.

[0008] The technical solution of this invention is: a method for gradient low-temperature acclimatization and constant low-temperature storage of adult bugs, comprising the following steps:

[0009] (1) Select 10-15 day old adult stink bugs and put them into a storage box containing a substrate and sticky bugs;

[0010] (2) Place the storage box into the artificial climate chamber for gradient low temperature acclimatization. The gradient low temperature acclimatization procedure is as follows: 26℃→20℃ acclimatization for 8h→15℃ acclimatization for 8h→10℃ acclimatization for 8h→6℃ acclimatization for 8h→4℃ acclimatization for 8h.

[0011] (3) After domestication, the adult bugs are placed at 4℃ for refrigeration for 40-60 days.

[0012] Preferably, the storage box contains sticky insects.

[0013] Preferably, the substrate is 20-40 mm of Danish Pindl peat moss.

[0014] Preferably, in step (3), the refrigeration time is 49 days, or 7 weeks.

[0015] Preferably, after refrigeration, the food is taken out and brought back to a normal temperature of 26°C, and fed honey water within 24 hours.

[0016] The beneficial effects of this invention are: by placing adult stink bugs into a storage box containing substrate and sticky bugs for gradient low-temperature acclimatization, and then storing them at 4°C for 40-60 days, this invention can ensure that they have normal oviposition capacity and egg hatching rate, as well as better offspring development and offspring survival rate during their lifespan.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] (1) By simulating the characteristics of winter climate change in nature, the bugs are subjected to gradient low temperature domestication and low temperature refrigeration. This can effectively improve the bugs' gradual adaptation to low temperature, so as to avoid them dying from sudden low temperature or causing damage to their reproductive function.

[0019] (2) By simulating the overwintering site of the bug in nature, 20-40mm Danish Pinscher peat moss was used in the storage box as the overwintering site for the bug to prevent self-harm. A certain number of armyworms were also placed in the storage box to prevent self-harm. This can effectively improve the survival rate of the bug.

[0020] (3) After refrigeration, the activity level of the army bugs is low. In order to prevent them from being preyed upon by the army bugs, they need to be allowed to recover at a normal temperature of 26℃ for 24 hours and fed honey water. After that, feed them with young army bugs for 2 days, and then feed them with older army bugs. Attached Figure Description

[0021] Figure 1 The effect of different low-temperature refrigeration times on the survival rate of adult stink bugs. Detailed Implementation

[0022] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0023] Implementation method:

[0024] This invention discloses a method for the gradient low-temperature acclimatization and constant low-temperature storage of adult crickets, comprising the following steps:

[0025] (1) Select 10-15 day old adult stink bugs and place them in a storage box containing substrate and 20 fifth-instar armyworms. If the selected stink bugs are too young, their ovaries and other developments will be poor, resulting in low egg production after storage and recovery. If they are too old, the stink bugs will have already started laying eggs in large quantities, making storage less meaningful. The substrate is 20-40mm Danish Pinscher peat moss, mainly composed of 20-40mm peat moss and a small amount of clay. This auxiliary material is moderately soft and has good air permeability, allowing stink bugs to move freely. After entering a low-temperature environment, they can easily burrow into the soil surface and remain still, which helps with low-temperature acclimatization and preservation. Place some armyworms in the storage box to ensure the adult stink bugs have enough to eat at room temperature and prevent them from self-cannibalizing, which can effectively improve the survival rate of the stink bugs.

[0026] (2) Place the storage box into the artificial climate chamber for gradient low temperature acclimatization. The gradient low temperature acclimatization procedure is as follows: 26℃→20℃ acclimatization for 8h→15℃ acclimatization for 8h→10℃ acclimatization for 8h→6℃ acclimatization for 8h→4℃ acclimatization for 8h.

[0027] (3) After domestication, place the adult bugs at 4℃ for 40-60 days. After refrigeration, take them out and restore them to the normal temperature of 26℃. Feed them honey water within 24 hours to prevent them from being preyed upon by the armyworms due to insufficient vitality.

[0028] Verification experiment:

[0029] To screen suitable constant low temperature storage and evaluate the survival rate, reproductive capacity, predation ability, offspring development, and offspring survival rate of adult stink bugs after storage, the experiment set up a control group (conventional rearing temperature 26℃) and three treatment groups (different constant low temperature storage temperatures: 0℃, 4℃, and 8℃). The results were compared under different treatment durations. The treatment methods for stink bugs before refrigeration were the same.

[0030] 1. Survival rate and egg production

[0031] Depend on Figure 1 It was observed that when the *Hemiberlesia lataniae* were refrigerated at 4℃ for 2, 4, and 6 weeks, their survival rate was 100%, and after 7 weeks of refrigeration, the survival rate was 91.67%, which was better than that at 0℃ and 8℃. After refrigeration, the *Hemiberlesia lataniae* were restored to the normal rearing temperature of 26℃. The fully recovered *Hemiberlesia lataniae* were then paired 1:1 and placed in disposable plastic cups with a 7cm×5cm piece of paper inside for egg laying. Fifth-instar armyworms were fed during this period, and the survival rate and egg production were recorded daily. The results showed that with the extension of the recovery time after refrigeration, both the treatment groups and the control group exhibited a decreasing trend in the normal survival rate during insect development. Compared with the control, the 4℃ refrigeration condition had the least impact on the survival rate of the *Hemiberlesia lataniae* after recovery. Figure 1After being refrigerated at 4℃ for 2, 4, 6, and 7 weeks, the total number of eggs laid by a single female of the cricket was 88.08%, 78.48%, 72.84%, and 60.37% of that of the control group, respectively, which could meet the needs of subsequent propagation (Table 1).

[0032] Table 1. Effects of different durations of 4℃ refrigeration on the egg production of *Aegilops chinensis* after recovery.

[0033]

[0034] 2. Egg hatching rate

[0035] After being refrigerated at 0℃, 4℃, and 8℃ for different periods, 100 eggs laid on the same day were collected from each insect. Egg hatching was observed and recorded daily, with three biological replicates. The results showed that the hatching rate of insect eggs refrigerated at 4℃ was significantly higher than that at 0℃ and 8℃. After two weeks of refrigeration, the hatching rate was lower than that of the control group. With extended refrigeration times of 4, 6, and 7 weeks, the hatching rate was comparable to the control (Table 2).

[0036] Table 2. Effects of low-temperature refrigeration on the hatching rate of *Aegilops spp.* eggs.

[0037]

[0038] Note: Different letters in the table indicate significant differences between treatment temperatures (p<0.05).

[0039] 3. Offspring development and offspring survival rate

[0040] The results of comprehensive analysis of survival rate, egg production, and hatching rate showed that 4℃ refrigeration had the least impact on the survival rate and egg production of adult stink bugs. Therefore, 4℃ was selected as the refrigeration condition to further evaluate offspring development and survival rate. After being refrigerated at 4℃ for different periods, the stink bugs were restored to normal rearing temperatures to induce egg production. One hundred eggs laid on the same day were collected, and the developmental period and survival rate of the offspring were observed and statistically analyzed. The results showed that after 2, 4, and 7 weeks of refrigeration, the developmental period of the offspring generations was not significantly different from the control, but it was slightly prolonged after 6 weeks of refrigeration (Table 3). After 2 and 4 weeks of refrigeration, the cumulative survival rate of the offspring was not significantly different from the control, but it slightly decreased after 6 weeks of refrigeration and increased after 7 weeks of refrigeration (Table 4).

[0041] Table 3. Effects of different refrigeration times at 4℃ on the developmental period of *Aegilops chinensis* offspring.

[0042]

[0043]

[0044] Note: Different letters in the table indicate significant differences between treatment times (p<0.05).

[0045] Table 4. Effects of different refrigeration times at 4℃ on the survival rate of *Aegilops chinensis* offspring.

[0046]

[0047] Note: Different letters in the table indicate significant differences between treatment times (p<0.05).

[0048] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for gradient low-temperature acclimatization and constant low-temperature storage of adult bugs, characterized in that, Includes the following steps: (1) Select 10-15 day old adult stink bugs and put them into a storage box containing a substrate and sticky bugs; (2) Place the storage box into the artificial climate chamber for gradient low temperature acclimatization. The gradient low temperature acclimatization procedure is as follows: 26℃→20℃ acclimatization for 8h→15℃ acclimatization for 8h→10℃ acclimatization for 8h→6℃ acclimatization for 8h→4℃ acclimatization for 8h. (3) After domestication, place the adult bugs at 4℃ for 40-60 days. After refrigeration, take them out and restore them to the normal temperature of 26℃. Feed them honey water within 24 hours.

2. The method for gradient low-temperature acclimatization and constant low-temperature storage of adult stink bugs according to claim 1, characterized in that, The storage box contained sticky insects.

3. The method for gradient low-temperature acclimatization and constant low-temperature storage of adult stink bugs according to claim 1, characterized in that, The substrate is 20-40 mm of Danish Pindt peat moss.

4. The method for gradient low-temperature acclimatization and constant low-temperature storage of adult stink bugs according to claim 1, characterized in that, In step (3), the refrigeration time is 49 days.

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