A method for verifying the effect of stress from Oriental flower stink bug on the activity of endogenous substances in soybean aphids

Abstract

A method for verifying that the stress of East Asian flower stink bug affects the activity of endogenous substances in soybean aphids, belongs to the field of agricultural science and technology, and is characterized by: the results described: compared with soybean aphids of the same age in the control group that are not stressed, the specific activity of acetylcholinesterase and carboxylesterase in the 1-4th instar soybean aphids stressed by East Asian flower stink bugs is significantly improved, and the maximum value is reached at the first instar. The specific activity of glutathione-S-transferase in the stressed 3-5th instar soybean aphids is lower than that in the treatment group, while the specific activity of the stressed 1-2th instar is significantly higher than that in the control group, and the specific activity reaches the maximum value at the second instar. The total protein content of the stressed 1-4th instar soybean aphids is significantly lower than that in the control group, while there is no significant difference between the stressed last instar soybean aphids and the control group. Conclusion: The stress of East Asian flower stink bugs has a significant effect on the content of various biochemical substances in the body of soybean aphids at young ages, but has little effect on the content of biochemical substances in the body of last instar soybean aphids.

Description

Technical Field

[0001] The invention relates to a method for verifying that the stress of oriental flower stink bug affects the activity of endogenous substances in soybean aphids, and belongs to the field of agricultural science and technology. Background Art

[0002] The East Asian flower bug (Orius sauteri) belongs to the family Anthozoidae of the order Hemiptera and is one of the important predatory natural enemy insects in northern my country. It has the characteristics of early occurrence, long duration, wide distribution, large number, miscellaneous diet, and strong activity (Yang Shufei et al., 2009). It mainly feeds on small insects such as thrips, aphids, spider mites, whiteflies, plant pollen, and eggs and small larvae of noctuid insects (Nagayi K, 1997). It plays an important role in controlling the occurrence of insect pests in orchards, farmlands, greenhouse flowers and trees, and has become the main species of insect natural enemy development and utilization. Domestic research has been conducted on the biological characteristics, occurrence, growth and decline, activity patterns and artificial breeding of the East Asian flower stink bug (Xu XN and Enkegaard A, 2009; Wu Yuqing et al., 2010; Huo Jie et al., 2011; Liu Wenjing et al., 2011; Huang Zengyu et al., 2011; Zhi Junrui et al., 2011; Jiang Xingchuan et al., 2012; Zhang Changrong et al., 2012; Ma Guangmin et al., 2012; Wang Hongliang et al., 2013), but there are few reports on its release and control effects (Jiang Yueli et al., 2011; Sun Meng et al., 2012). As one of the main pests of soybean in the field, soybean aphid can also cause a variety of viral diseases mediated by it. If it is not controlled in time, it will bring inestimable losses. Giving full play to the role of natural enemy resources is an important link in establishing a soybean aphid prevention and control system. In indoor studies, the East Asian flower stink bug has shown strong predation and control potential on soybean aphids. Predator-prey interactions are at the core of biological control of arthropod pests and have important application value in fields such as agricultural pest management and natural resource conservation (Tscharntke T. et al., 2007; Straub C Set al., 2008; Zehnder G. et al., 2007; De Clercq P. et al., 2011; Lu Y. et al., 2012; Attia S. et al., 2013). Reduced costs for labor, pesticides, and equipment, as well as a possible return to pre-invasion ecological services, constitute a persuasive argument for strengthening conservation biological control in the coming years (Liu YQ. et al., 2014; Pickett C H. and Bugg RL, 1998; Landis D A. et al., 2000). When Bokonon-Ganta evaluated the economic benefits of biological control of mango mealybugs, they found that the benefit-cost ratio was 145:1 (Bokonon-Ganta. et al., 2002; DeGroote H. et al., 2003).This does not yet measure the costs of some unsuccessful biological control programs (Cortesero A. et al., 2000; Altieri M A. and Nicholls C I., 2004; Messing R H. and Wright M G., 2006; Jonsson M. et al., 2008). However, attention should be paid to the possibility of interactions with non-target organisms (Howarth F G., 1991; Louda S M. et al., 2013). To reduce or avoid such interactions, new tools have been developed, including host-specific experiments conducted in target habitats whenever possible (Desneux N. et al., 2009; Desneux N., 2012). Today, a large number of predatory arthropods are collected and mass-raised and then released in target agricultural ecosystems to enhance biological control programs (Pickett CH. and Bugg R L., 1998; Symondson W., 2002). These programs benefit from tools that can be used to attract, collect and monitor predatory arthropods in natural habitats and target crops. There are many kinds of natural enemies of soybean aphids in nature. Dai Changchun, Wang Kun and others analyzed the community structure of natural enemy insects in soybean aphid fields and found that in the early stage of soybean aphid occurrence, a small number of small flower bugs, tortoise-patterned ladybugs, leaf-colored lacewings and predatory spiders were active in the fields, which delayed the approach of the soybean aphid outbreak to a certain extent and reduced its initial occurrence. Most of the dominant natural enemy populations, such as Harmonia axyridis, Chinese lacewings, black aphid-eating blind bugs, narrow-headed assassin bugs, and black-banded aphid flies, appeared when the number of soybean aphids was large, playing a certain role in controlling the pest during the peak period of soybean aphid occurrence. However, aphid wasps and aphid gall midges occurred in large numbers in the late stage of soybean aphid occurrence, reducing the base number of insects migrating back to the soybean fields the next year and suppressing the insect source (Dai Changchun et al., 2009). For agricultural ecosystems, it is necessary to identify pests and natural enemy resources, master the structure and characteristics of the community, understand the structure and dynamics of the insect community in the ecosystem, take the farmland ecosystem as the regulatory unit, and give full play to the natural control force from the perspective of the community, that is, to make full use of the mutual promotion and mutual restriction relationship between organisms and the non-biological environment to regulate the population of harmful organisms to prevent them from causing economic damage. This is the basis for maintaining natural balance and controlling harmful organisms (Wratten, 1987; Ding Yanqin, 1993; Ge Feng, 1998). Using the regulatory role of predatory natural enemy insects in nature, using insects to control insects and controlling insect disasters is an effective way to ensure agricultural production and protect a clean agricultural environment. It is also conducive to achieving agricultural ecological balance, protecting biodiversity and achieving the lofty goal of harmonious coexistence between humans and various organisms.As a natural enemy resource with great utilization value, the East Asian flower stink bug has a good control and regulation effect on some small pests such as aphids, thrips and whiteflies, as well as the eggs and larvae of noctuid insects on crops such as cotton, corn, sorghum, soybeans and various vegetables (Dong Tianyu et al., 2015). As a natural enemy of soybean aphids, its existence will definitely have a certain coercive effect on soybean aphids. After visual impact, odor stimulation and direct contact, soybean aphids will produce certain fluctuations in the content of substances in the body. The generation of this fluctuation must be related to the production and changes of various substances involved in respiration, nerve transmission, detoxification ability and self-protection functions in the body. The change of a substance will inevitably lead to changes in related substances in order to maintain the constancy in the body. For example, when acetylcholine is increased after the organism is stimulated, in order to maintain the safety of the body, the content of esterase that decomposes acetylcholine in the body will also increase, so that the organism maintains a relative balance to a certain extent, protecting the soybean aphid from damage due to excessive changes in a substance. This mechanism is often called a self-protection mechanism, which exists in various organisms. In the present invention, by measuring and analyzing the changes in various physiological indicators in soybean aphids of different ages after being stressed by the East Asian flower stink bug, the relationship between the content of different biochemical substances and the changes in the activity of protective enzymes in soybean aphids of different ages and the defense mechanism of soybean aphids under the stress of the East Asian flower stink bug was preliminarily explored, which provides a theoretical basis for studying the stress mechanism and comprehensive management of the East Asian flower stink bug. Trehalose is the main blood sugar in insects. Trehalose has biological properties different from other carbohydrates and can protect components such as proteins, fats, sugars, and nucleic acids in biological cells from damage in harsh environments. Under harsh environmental conditions, a unique protective film can be formed on the cell surface, effectively protecting the protein molecules from inactivation, thereby maintaining the life process and biological characteristics of the organism (Crowe et al., 1990; Williams, 1990; Cheng Yuan, 2016). In the present invention, after using the East Asian flower stink bug to stress soybean aphids of different ages in various ways, the trehalose content in the soybean aphids of each age is significantly higher than that in the control group, which indicates that after being stressed, the self-protection mechanism of soybean aphids is activated, resulting in a significant increase in the synthesis of the protective substance trehalose in the soybean aphids. It is theoretically proved that the stress of the East Asian flower stink bug can effectively stimulate the soybean aphid to start the defense mechanism, thereby causing the soybean aphid to be in a state of stress in advance, and the physical fitness declines after a period of time, causing the defense mechanism of soybean aphids to decline, which is similar to the research results of the effect of the parasitism of the light yellow aphid wasp on the trehalose content of Bemisia tabaci (Wang Yingying et al., 2015). In general, the effect of direct stress on the trehalose content in soybean aphids is more significant, while the significance of visual stress decreases with the decrease of age. This shows that the trehalose content of soybean aphids is more significantly affected by the visual stress of the East Asian flower stink bug at an advanced age, which may be due to the relatively complete development of adult aphids, so the ability to perceive visual stress is stronger.However, it may also be because after 24 hours of stress treatment, the trehalose content in the young soybean aphids was first affected by trehalase and restored to a more stable state than that in the older soybean aphids, and the proteins and genes that affect synthesis or dissociation need further research. Reducing sugars mainly include glucose, fructose and maltose, among which glucose and fructose are the main substrates of respiration. Therefore, after the stress of the East Asian flower stink bug, the respiration in the soybean aphid was enhanced, and the substrates required for glycolysis needed to increase significantly to be consumed by respiration. Therefore, after the stress, the reducing sugar content in the soybean aphid increased significantly, but the reducing sugar content in the adult aphid stage increased slightly, but there was no significant difference at the 0.05 level. The reason may be that the stress on the adult aphids did not have much effect on their respiration, while the impact on the young soybean aphids was significantly different at the 0.01 level, which shows that the stress of the East Asian flower stink bug has a significant effect on the young soybean aphids. In general, the contact stress and odor stress of the East Asian flower stink bug have more significant effects on soybean aphids, which indicates that the early-stage soybean aphids are more significantly affected by odor stress than by visual stress. There are many kinds of proteins in insects, which are not only related to the material metabolism of tissue formation, but also closely related to the hormone regulation of insect growth and development, as well as the generation of drug resistance and immune mechanisms (Sun Haoyu et al., 2014). In the present invention, visual impact, odor perception stimulation and direct contact are used to treat soybean aphids of various ages. The results show that the protein content in soybean aphids of various ages after being stressed by the East Asian flower stink bug is significantly lower than that in the control group. This may be due to the synthesis of various enzymes and other substances in the body of soybean aphids and the consumption of respiratory systems after being affected by stress. From the perspective of age, the stress caused by visual impact has a more significant effect on the protein content in the body of soybean aphids at high ages, while at low ages, the stress caused by direct contact between the East Asian flower stink bug and soybean aphid has a more significant effect on the protein content in the body of soybean aphids. This may be because the visual system of soybean aphids is more fully developed in the adult stage, and because of the predatory nature of the East Asian flower stink bug, it prefers to prey on soybean aphids at low ages, so the contact may not have as significant an effect on soybean aphids at high ages as on those at low ages. In terms of the differences between the stress methods, [direct stress has a more thorough effect (large changes), while the effects of odor and vision are relatively stable]. Such changes in protein will inevitably lead to changes in enzyme substances, and further research should be done on the proteins and genes that affect synthesis or dissociation. The protective enzymes in insects work in coordination with each other to protect the balance of the insect body and defend against damage to the organism by adverse conditions and substances. Changes in the activity of host protection enzymes can be used as an important indicator to determine the strength of external stimuli and the insect's resistance to pesticides and stress (Ren Xiaoya, 2014; Wu Haibin, 2018). Soybean aphids produce a variety of stress defense response mechanisms after being threatened by the East Asian flower bug, and enzymes are the basis of these reactions. Among them, AchE, CarE and GSTs are important detoxification and protection enzymes.Acetylcholinesterase belongs to serine hydrolase and is widely present in various animal tissues and serum. Acetylcholinesterase catalyzes the hydrolysis of acetylcholine and plays a regulatory role in nerve conduction. In the present invention, after soybean aphids of various ages were subjected to the stress of the East Asian flower stink bug, the specific activity of acetylcholinesterase of soybean aphids of advanced ages was significantly different from that of the control group, but the specific activity of acetylcholinesterase of soybean aphids of lower ages was much different from that of the control group. This shows that the stress of the East Asian flower stink bug is more significant to soybean aphids of lower ages. Moreover, the stress caused by odor and direct contact is a more significant factor in stimulating soybean aphids of lower ages. Carboxylesterase participates in the transport and metabolism of lipids, and is related to the detoxification and metabolism of various drugs and poisons. Whether it is affected or not is an important influencing factor for the comprehensive control of soybean aphids such as biological control and chemical control. Therefore, how to verify that the stress of East Asian flower stink bug affects the activity of endogenous substances in soybean aphids has become a major problem that needs to be solved urgently. The East Asian flower stink bug was used to stress soybean aphids. After experiments, it can be seen that the stress of East Asian flower stink bug has a significant effect on the carboxylesterase specific activity of soybean aphids. It was also found that the carboxylesterase specific activity of high-age soybean aphids composed of 3rd, 4th and adult aphids was more significantly affected by the visual stress of East Asian flower stink bugs, while the carboxylesterase specific activity of low-age soybean aphids composed of 1st and 2nd instars was more significantly affected by the odor stress of East Asian flower stink bugs. The reason may be that the vision of high-age soybean aphids is well developed, while the vision of low-age soybean aphids is not well developed, so they rely more on the antennae to recognize the odor, and the impact caused by the odor stress of East Asian flower stink bugs is more obvious. Glutathione-S-transferase is an important component of the detoxification enzyme system in the body. It mainly catalyzes the covalent binding of various chemical substances and their metabolites to the sulfhydryl group of GSH, converting electrophilic compounds into hydrophilic substances, so as to achieve the purpose of degrading and excreting various potential or toxic substances in the body. Therefore, the specific activity of glutathione-S-transferase in soybean aphids after being stressed by the East Asian flower bug is roughly the same as that of carboxylesterase. The older soybean aphids composed of stressed adult aphids and 4-instar nymphs are more significantly affected by the visual stress of the East Asian flower bug, while the younger soybean aphids composed of 3-instar, 2-instar and 1-instar nymphs are more significantly affected by the stress caused by the odor stress and direct contact of the East Asian flower bug. It is necessary to invent a method to verify that the stress of the East Asian flower bug affects the activity of endogenous substances in soybean aphids. Summary of the invention

[0003] In order to overcome the problem of how to verify that the stress of East Asian flower stink bug affects the vitality of endogenous substances in soybean aphids, the present invention provides a method for verifying that the stress of East Asian flower stink bug affects the vitality of endogenous substances in soybean aphids. The method for verifying that the stress of East Asian flower stink bug affects the vitality of endogenous substances in soybean aphids uses East Asian flower stink bug to stress soybean aphids. After experiments, it can be seen that the stress of East Asian flower stink bug has a significant effect on the carboxylesterase specific activity of soybean aphids. It is also found that the carboxylesterase specific activity of high-age soybean aphids composed of 3rd, 4th and adult aphids is more significantly affected by the visual stress of East Asian flower stink bugs, while the carboxylesterase specific activity of low-age soybean aphids composed of 1st and 2nd ages is more significantly affected by the odor stress of East Asian flower stink bugs. The reason may be that the vision of high-age soybean aphids is well developed, while the vision of low-age soybean aphids is not well developed, so they rely more on the recognition of odors by antennae, so the impact caused by the odor stress of East Asian flower stink bugs is more obvious. Glutathione-S-transferase is an important component of the detoxification enzyme system in the body. It mainly catalyzes the covalent binding of various chemical substances and their metabolites to the thiol group of GSH, turning electrophilic compounds into hydrophilic substances, and achieving the purpose of degrading and excreting various potential or toxic substances in the body. Therefore, the specific activity of glutathione-S-transferase in soybean aphids after being stressed by the East Asian flower bug is roughly the same as that of carboxylesterase. The older soybean aphids, consisting of stressed adult aphids and 4th-instar nymphs, are more significantly affected by the visual stress of the East Asian flower bug, while the younger soybean aphids, consisting of 3rd-instar, 2nd-instar and 1st-instar nymphs, are more significantly affected by the odor stress and direct contact stress of the East Asian flower bug.

[0004] The technical solution adopted by the present invention to solve its technical problem is:

[0005] The present invention provides a method for verifying that the stress of Ornithogalum spp. affects the activity of endogenous substances in soybean aphids, and the specific experimental steps are as follows:

[0006] 1 Materials and methods

[0007] 1.1 Test materials and instruments

[0008] 1.1.1 Test insects

[0009] Orius sauteri was collected from the soybean field of Xiangyang Experimental Farm in Heilongjiang Province, raised in a constant temperature incubator (temperature T = 25 ± 1 ° C, relative humidity RH = 70% ± 5%, light L: D = 16: 8), fed with soybean aphids, and bred with soybean sprouts as ovipositors.

[0010] The soybean aphid Aphis glycines is a local sensitive population of Harbin that has been preserved in the laboratory for a long time. It was raised in artificial potted soybean seedlings and placed in an artificial climate box with a temperature of 25±1℃, a relative humidity of 65%-70%, and a photoperiod of 14L:10D. The tested soybean variety was Dongnong 52.

[0011] 1.1.2 Experimental reagents

[0012] Coomassie brilliant blue G-250 and bovine serum albumin (BSA) were purchased from Beijing TaKaRa Biotechnology Co., Ltd. Phosphate buffer (PB) was purchased from Shanghai Sangon Biotechnology Co., Ltd. Acetylcholinesterase (AchE), carboxylesterase (CarE), glutathione-S-transferase (GSTs), trehalose, and reducing sugar were purchased from Beijing Solarbio Technology Co., Ltd.

[0013] 1.1.3 Instruments and Equipment

[0014] High-speed low-temperature refrigerated centrifuge (ThermoSorvallST16R), artificial climate chamber (BIC, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), multifunctional microplate reader (INFINITE-200PRO, Swiss TECAN Company), oven (DPH781, Hubei Huangshi Medical Equipment Factory), refrigerator (Meiling), electric constant temperature water bath (DZKW-S-4, Beijing Yongguangming Medical Instrument Factory), quartz sand (analytical grade, Tianjin BASF Chemical Co., Ltd.), anhydrous ethanol (Tianjin Tianli Chemical Reagent Co., Ltd.)

[0015] 1.2 Stress treatment of soybean aphid by Orientia spp.

[0016] The soybean aphids of various ages were subjected to stress treatment with adult East Asian flower stink bugs. The treatment methods were divided into three types: contact stress, odor stress and visual stress. The control group was not subjected to East Asian flower stink bug stress. A certain number of soybean aphids were placed on the upper layer of each homemade culture dish, and the adult East Asian flower stink bugs were placed on the lower layer. The middle of the culture dish was isolated by a transparent visible plastic plate and an invisible air filter with pinholes. After treatment, it was placed in an incubator with a temperature of 24±1℃, a relative humidity of 70%±5%, and a light intensity of 16L:8D. After 24h, the stressed soybean aphids were taken for various biochemical substances.

[0017] 1.3 Enzyme solution preparation

[0018] Referring to the enzyme extraction method of Gao Huanhuan et al. (2016), after 24 hours, 5 instars of soybean aphids were selected in each treatment, and 5 surviving insects of each instar were collected and placed in a 1.5mL Eppendorf centrifuge tube. 400μL 0.02mol / L phosphate buffer (NaCl 136.89mM; KCl 2.67mM; Na2HPO4 8.1mM; KH2PO4 1.76mM) and a small amount of quartz sand were added, and the mixture was fully ground in an ice bath, and the ground homogenate was placed in a high-speed refrigerated centrifuge at 8000g and 4℃ for 10min. The supernatant was divided into a new 1.5mL Eppendorf centrifuge tube and placed on ice for testing. Three replicates were set for the 5 instars of each treatment.

[0019] 1.4 Determination of protein, reducing sugar and trehalose content

[0020] 1.4.1 Determination of protein content

[0021] The determination was carried out with reference to the Coomassie Brilliant Blue G-250 staining method of Bradford (1976). Coomassie Brilliant Blue G-250 solution and bovine serum albumin standard solution were prepared respectively to prepare a standard curve. 4 μL of enzyme solution was added to the sample well of the ELISA plate for each sample, 4 μL of phosphate buffer was added to the control well, and then 200 μL of rewarmed Coomassie Brilliant Blue G-250 reagent was added, mixed thoroughly, and placed at room temperature for 5 minutes. The ELISA reader was set at a wavelength of 595 nm for determination, and the OD value was recorded. Repeat 3 times, and the protein concentration in each sample to be tested was calculated according to the protein standard curve.

[0022] 1.4.2 Determination of reducing sugar and trehalose content

[0023] The content of trehalose was determined by the anthrone sulfuric acid method. Anthrone solution and 10 mg / mL trehalose standard solution were prepared to prepare a standard curve. For each sample, 60 μL of sample solution and 240 μL of prepared anthrone reagent were mixed in a 1.5 mL Eppendorf centrifuge tube, 95°C water bath for 10 min (cover tightly to prevent water loss), and naturally cooled to room temperature. 200 μL was taken to the ELISA plate, and the measurement was performed at a wavelength of 620 nm. The OD value was recorded and repeated 3 times. According to the trehalose standard curve, the trehalose content in each sample to be tested was calculated.

[0024] The content of reducing sugar was determined by DNS method. 3,5-dinitrosalicylic acid (DNS) alkaline solution and 10 mg / mL glucose standard solution were prepared to prepare standard curve. In the test tube, 175 μL sample solution and 125 μL prepared DNS reagent were mixed in a 1.5 mL Eppendorf centrifuge tube for each sample, heated in a boiling water bath for 5 min (covered tightly to prevent water loss), and immediately cooled to room temperature and mixed. In the control tube, 175 μL sample solution and 125 μL distilled water were mixed in a 1.5 mL Eppendorf centrifuge tube for each sample, heated in a boiling water bath for 5 min (covered tightly to prevent water loss), and immediately cooled to room temperature and mixed. Take 200 μL to a 96-well ELISA plate, measure at a wavelength of 540 nm, record the OD value, and repeat 3 times. The difference in absorbance between the test tube and the control tube was calculated, and the reducing sugar content in each sample was calculated based on the glucose standard curve (Wu Junxiang et al., 2004; Ding Huimei, 2011; Cheng Yuan et al., 2016).

[0025] 1.5 Determination of enzyme activity

[0026] The determination of the specific activity of AchE, CarE and GSTs enzymes was carried out according to the instructions in the kit of Beijing Solebow Technology Co., Ltd. The enzyme activity value was calculated according to the formula in the kit manual. The wavelengths for the determination of the specific activity of AChE, CarE and GSTs enzymes were 412, 450 and 340 nm, respectively. The AchE activity unit is defined as the production of 1 nmol of molar extinction coefficient (TNB) per minute per milligram of tissue protein as one unit of enzyme activity; the CarE activity unit is defined as the increase of 1 catalytic absorbance per minute per milliliter of reaction system per milligram of tissue protein at 37°C as one unit of enzyme activity; the GST activity unit is defined as the catalysis of 1 μmol of 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) per minute per milligram of protein at 25°C or 37°C as one unit of enzyme activity.

[0027] 1.6 Data Analysis

[0028] All experimental data were analyzed and plotted using Microsoft Excel and SPSS Base Ver.19.0, and Duncan's new multiple range test was used to analyze the differences among treatments. One-way ANOVA was used to analyze the protein, trehalose and reducing sugar contents and the activities of AchE, CarE and GSTs in soybean aphids after being stressed by Orientia spp.

[0029] 2 Results

[0030] 2.1 Effects of Ornithogalum spp. stress on protein content in soybean aphids

[0031] After 24 hours of stress from the Oriental flower bug to soybean aphids, the protein content of the enzyme solution showed an increasing trend as the age of the aphids increased. Figure 1 The results showed that compared with the control group, at the adult aphid stage, the protein content of enzyme solution in the odor stress and visual stress treatment groups decreased by 23.42% and 57.27%, respectively, and the difference between the visual stress treatment group and other groups was extremely significant, while there was no significant difference between the direct contact stress and the control group; at the 4th instar, compared with the control group, the protein content of the odor stress and visual stress treatment groups decreased by 46.36% and 61.85%, respectively, while the direct contact stress decreased by 44.33%; at the 3rd instar, the protein content of the odor stress and visual stress treatment groups decreased by 27.99% and 30.15%, respectively, while the direct contact stress decreased by 3. 1.54%, and there was no significant difference among the three treatment groups; at the 2nd age, the protein content of the odor stress and visual stress treatment groups decreased by 15.82% and 24.64%, respectively, and that of direct contact stress decreased by 52.73%; at the 1st age, the protein content of the odor stress, visual stress and direct contact stress treatment groups decreased by 15.13%, 33.32% and 57.01%, respectively, and the protein content in the direct contact stress treatment group reached the lowest value (9.12 μg / mL), which was significantly lower than that of the control group, odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0032] 2.2 Effects of Ornithogalum spp. stress on trehalose content in soybean aphids

[0033] After 24 hours of stress from the Oriental flower stink bug to soybean aphid, the trehalose content in the stink bug showed an increasing trend as the age increased. Figure 2 The results showed that compared with the control group, at the adult aphid stage, the trehalose content in the odor stress and visual stress treatment groups increased by 97.77% and 43.49%, respectively, and the direct contact stress treatment group reached the highest value (3.4607 mg / mgprot), and there were extremely significant differences between the three stress methods and the control group; at the 4th instar, the trehalose content in the odor stress and visual stress treatment groups increased by 67.08% and 32.56%, respectively, and the direct contact stress increased by 104.12%; at the 3rd instar, the odor stress treatment group increased by 104.12%, and the visual stress treatment group increased by 104.12%. The trehalose content of the odor stress and visual stress treatment groups increased by 52.29% and 22.22% respectively, and the direct contact stress increased by 115.33%; at the 2nd instar, the trehalose content of the odor stress and visual stress treatment groups increased by 72.21% and 30.22% respectively, and the direct contact stress increased by 145.48%; at the 1st instar, the odor stress, visual stress and direct contact stress treatment groups increased by 57.24%, 37.96% and 160.72% respectively, and there was no significant difference between the odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0034] 2.3 Effects of Ornithogalum spp. stress on reducing sugar content in soybean aphids

[0035] After 24 hours of stress from the Oriental flower stink bug to soybean aphid, the reducing sugar content of the aphid showed a decreasing trend as the age increased. Figure 3 The results showed that compared with the control group, at the adult aphid stage, there was no significant difference in reducing sugar content between the odor stress, visual stress and direct contact stress treatment groups and the control group; at the 4th instar, the reducing sugar content of the odor stress and visual stress treatment groups increased by 27.29% and 11.36%, respectively, and that of direct contact stress increased by 46.10%, and the reducing sugar content of the three treatment groups was significantly higher than that of the control group, while that of the direct contact stress treatment group was extremely significantly higher than that of the odor stress and visual stress treatment groups; at the 3rd instar, the reducing sugar content of the odor stress and visual stress treatment groups increased by 69.72% and 55.27%, respectively, and that of direct contact stress increased by 120%, and the odor stress treatment group increased by 120%. There was no significant difference between the stress and visual stress treatment groups; at the second age, the reducing sugar content of the odor stress and visual stress treatment groups increased by 122.67% and 114.88%, respectively, and that of direct contact stress increased by 168.73%; at the first age, the reducing sugar content of the odor stress, visual stress and direct contact stress treatment groups increased by 308.95%, 193.53% and 371.54%, respectively, and the reducing sugar content in the direct contact stress treatment group reached the highest value (2.89 μg / mgprot), which was significantly higher than that of the control group, odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0036] 2.4 Effects of Ornithogalum spp. stress on the specific activity of acetylcholinesterase in soybean aphids

[0037] After 24 hours of stress from the Oriental flower stink bug to soybean aphid, the acetylcholinesterase activity of the stink bug showed a decreasing trend as the age of the stink bug increased. Figure 4The results showed that compared with the control group, at the adult aphid stage, the acetylcholinesterase activity in the odor stress and visual stress treatment groups decreased by 68.34% and 70.57%, respectively, and there was no significant difference between the odor stress and visual stress treatment groups, while there was no significant difference between the direct contact stress and the control group; at the 4th instar, the acetylcholinesterase activity in the odor stress and direct contact stress treatment groups increased by 56.91% and 104.57%, respectively, and the odor stress and direct contact stress treatment groups were significantly higher than the control group, while there was no significant difference between the visual stress treatment group and the control group; at the 3rd instar, the odor stress and visual stress treatment groups increased by The acetylcholinesterase activity in the odor stress and visual stress treatment groups increased by 368.71% and 136.04%, respectively, while that in the direct contact stress treatment groups increased by 918.05%. At the age of 1, the acetylcholinesterase activity in the odor stress, visual stress and direct contact stress treatment groups increased by 4228.37%, 1628.48% and 8506.98%, respectively, and the acetylcholinesterase activity in the direct contact stress treatment group reached the highest value (198.79 U / mg prot), which was significantly higher than that in the control group, odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0038] 2.5 Effects of Ornithogalum spp. stress on the specific activity of carboxylesterase in soybean aphids

[0039] After 24 hours of stress on soybean aphids by the Oriental flower stink bug, the specific activity of carboxylesterase showed a significant downward trend with the increase of age. Figure 5The results showed that compared with the control group, at the adult aphid stage, the carboxylesterase activity of the odor stress and visual stress treatment groups increased by 174.30% and 887.64%, respectively, and the visual stress and odor stress treatment groups were significantly higher than the control group, while there was no significant difference between the direct contact stress and the control group; at the 4th instar, the carboxylesterase activity of the odor stress and visual stress treatment groups increased by 126.16% and 586.81%, respectively, and the direct contact stress increased by 124.52%, and the three stress methods were significantly higher than the control group, while there was no significant difference between the odor stress and direct contact stress treatment groups; at the 3rd instar, the odor stress and visual stress treatment groups were significantly higher than the control group, while there was no significant difference between the odor stress and direct contact stress treatment groups. The activity of carboxylesterase in the odor stress and visual stress treatment groups increased by 55.48% and 1508.95% respectively, while that in direct contact stress increased by 173.87%, and there were extremely significant differences among the three treatment groups; at the 2nd age, the activity of carboxylesterase in the odor stress and visual stress treatment groups increased by 1135.72% and 875.35% respectively, while that in direct contact stress increased by 581.85%; at the 1st age, the activity of carboxylesterase in the odor stress, visual stress and direct contact stress treatment groups increased by 1889.46%, 357.15% and 1713.34% respectively, and the activity of carboxylesterase in the odor stress treatment group reached the highest value (0.0038U / mg prot), which was significantly higher than that in the control group, odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0040] 2.6 Effects of Ornithogalum spp. stress on the specific activity of glutathione-S-transferase in soybean aphids

[0041] After 24 hours of stress on soybean aphids by the Oriental flower stink bug, the specific activity of glutathione-S-transferase showed a significant downward trend with the increase of age. Figure 6The results showed that compared with the control group, at the adult aphid stage, the glutathione-S-transferase activity in the odor stress and visual stress treatment groups increased by 97.62% and 229.91%, respectively, while that in the direct contact stress treatment group decreased by 58.5%. The differences between the three treatment groups and the control group were extremely significant, while the glutathione-S-transferase activity in the direct contact stress treatment group was extremely significantly lower than that in the control group. At the 4th instar, the glutathione-S-transferase activity in the odor stress and visual stress treatment groups increased by 81.78% and 219.26%, respectively, while that in the direct contact stress treatment group decreased by 12.19%. There was no significant difference between the stress treatment group and the control group; at the 3rd instar, the odor stress and visual stress treatment groups increased by 20.55% and 10%, respectively, while the direct contact stress decreased by 58.45%; at the 2nd instar, the glutathione-S-transferase specific activity of the odor stress and visual stress treatment groups increased by 31.17% and 7.39%, respectively, while the direct contact stress increased by 156.59%, and the direct contact stress and odor stress treatment groups were significantly higher than the control group, and the glutathione-S-transferase specific activity in the direct contact stress treatment group reached the highest value (0.06nmol / min / mg prot), while there was no significant difference between the visual stress treatment group and the control group; at 1 year old, the odor stress, visual stress and direct contact stress treatment groups increased by 10.23%, 2.54% and 104.42%, respectively, and the direct stress was significantly higher than the control group, odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0042] The beneficial effect of the present invention is a method for verifying that the stress of East Asian flower stink bug affects the activity of endogenous substances in soybean aphids. The East Asian flower stink bug is used to stress soybean aphids. After experiments, it can be seen that the stress of East Asian flower stink bug has a significant effect on the carboxylesterase specific activity of soybean aphids. It is also found that the carboxylesterase specific activity of high-age soybean aphids composed of 3rd instar, 4th instar and adult aphids is more significantly affected by the visual stress of East Asian flower stink bugs, while the carboxylesterase specific activity of low-age soybean aphids composed of 1st instar and 2nd instar is more significantly affected by the odor stress of East Asian flower stink bugs. The reason may be that the vision of high-age soybean aphids is well developed, while the vision of low-age soybean aphids is not well developed, so they rely more on the recognition of odors by antennae, and thus the impact caused by the odor stress of East Asian flower stink bugs is more obvious. Glutathione-S-transferase is an important component of the detoxification enzyme system in the body, mainly catalyzing various chemical substances and their metabolites to covalently bind to the sulfhydryl group of GSH, so that electrophilic compounds become hydrophilic substances, and achieve the purpose of degrading various potential or toxic substances in the body and excreting them from the body. Therefore, the glutathione-S-transferase specific activity in the soybean aphid after being stressed by the East Asian flower stink bug shows a roughly similar situation to the carboxylesterase, and the high-age soybean aphid composed of the stressed adult aphid and the 4-year-old nymph aphid is more significantly affected by the visual stress of the East Asian flower stink bug, and the low-age soybean aphid composed of the 3-year-old, 2-year-old and 1-year-old nymph aphid is more significantly affected by the stress caused by the odor stress of the East Asian flower stink bug and direct contact. The present invention is simple to operate, easy to use, and has obvious effects. It has been verified that the East Asian flower stink bug stress affects the vitality of endogenous substances in the soybean aphid, and is an effective verification method. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The present invention is further described below in conjunction with the accompanying drawings.

[0044] Figure 1 The present invention is a method for verifying that the stress of the oriental flower bug affects the activity of endogenous substances in the soybean aphid, and a graph showing protein content values ​​at different ages of the soybean aphid after being stressed by the oriental flower bug.

[0045] Figure 2 The present invention provides a method for verifying that the stress of the oriental flower bug affects the activity of endogenous substances in the soybean aphid, and a graph showing changes in trehalose content at different ages of the soybean aphid after being stressed by the oriental flower bug.

[0046] Figure 3 The present invention is a method for verifying that the stress of the oriental flower bug affects the activity of endogenous substances in the soybean aphid, and is a graph showing changes in reducing sugar content in soybean aphids at different ages after being stressed by the oriental flower bug.

[0047] Figure 4 The present invention is a method for verifying that the stress of Ornithogalum spp. affects the activity of endogenous substances in soybean aphids, and is a graph showing changes in the acetylcholinesterase specific activity of soybean aphids at different ages after being stressed.

[0048] Figure 5 The present invention is a method for verifying that the stress of Ornithogalum spp. affects the activity of endogenous substances in soybean aphids. The present invention is a diagram showing the changes in the carboxylesterase specific activity of soybean aphids at different ages after being stressed.

[0049] Figure 6 The present invention is a method for verifying that the stress of Ornithogalum sphaerocephalus affects the activity of endogenous substances in soybean aphids, and a graph showing the changes in the relative activity of glutathione-S-transferase of soybean aphids at different ages after being stressed. DETAILED DESCRIPTION

[0050] Embodiment 1:

[0051] The present invention provides a method for verifying that the stress of Orius sauteri affects the activity of endogenous substances in soybean aphids. The purpose is that soybean aphid Aphis glycines is an important piercing-sucking pest that harms my country's soybean yield, and Orius sauteri is one of the main natural enemies of soybean aphids in Northeast my country. Clarifying the stress effect between Orius sauteri and soybean aphids will help us understand the response relationship between pests and natural enemies, explore the practical application value of Orius sauteri as a biological pesticide, guide precise drug use, and provide a scientific basis for integrated pest management. Methods: Under different stress conditions in the laboratory, the total protein content and the specific activities of acetylcholinesterase, carboxylesterase and glutathione-S-transferase in soybean aphids of different ages were determined. Results: Compared with the same-age soybean aphids in the control group without stress, the specific activities of acetylcholinesterase and carboxylesterase in 1-4 instar soybean aphids stressed by Orientia spp. increased significantly, and the specific activity of carboxylesterase reached a maximum of 0.0035U / mg prot in the first instar. However, the specific activities of acetylcholinesterase and carboxylesterase in the last instar soybean aphids under stress did not increase significantly compared with the control group. The specific activity of glutathione-S-transferase in 3-5 instar soybean aphids under stress was lower than that in the treatment group, while the specific activity of glutathione-S-transferase in 1-2 instar soybean aphids under stress was significantly higher than that in the control group, and the specific activity of glutathione-S-transferase reached a maximum of 0.1622nmol / min / mg prot in the second instar. The total protein content of 1-4 instar soybean aphids under stress was significantly lower than that in the control group, while the total protein content of the last instar soybean aphids under stress was not significantly different from that in the control group. Conclusion: The stress of Ornithogalum spp. has a significant effect on the contents of various biochemical substances in the early instar soybean aphids, but has little effect on the contents of biochemical substances in the late instar soybean aphids.

[0052] 2 Materials and methods

[0053] 1.1 Test materials and instruments

[0054] 1.1.1 Test insects

[0055] Orius sauteri was collected from the soybean field of Xiangyang Experimental Farm in Heilongjiang Province, raised in a constant temperature incubator (temperature T = 25 ± 1 ° C, relative humidity RH = 70% ± 5%, light L: D = 16: 8), fed with soybean aphids, and bred with soybean sprouts as ovipositors.

[0056] The soybean aphid Aphis glycines is a local sensitive population of Harbin that has been preserved in the laboratory for a long time. It was raised in artificial potted soybean seedlings and placed in an artificial climate box with a temperature of 25±1℃, a relative humidity of 65%-70%, and a photoperiod of 14L:10D. The tested soybean variety was Dongnong 52.

[0057] 1.1.2 Experimental reagents

[0058] Coomassie brilliant blue G-250 and bovine serum albumin (BSA) were purchased from Beijing TaKaRa Biotechnology Co., Ltd. Phosphate buffer (PB) was purchased from Shanghai Sangon Biotechnology Co., Ltd. Acetylcholinesterase (AchE), carboxylesterase (CarE), glutathione-S-transferase (GSTs), trehalose, and reducing sugar were purchased from Beijing Solarbio Technology Co., Ltd.

[0059] 1.1.3 Instruments and Equipment

[0060] High-speed low-temperature refrigerated centrifuge (ThermoSorvallST16R), artificial climate chamber (BIC, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), multifunctional microplate reader (INFINITE-200PRO, Swiss TECAN Company), oven (DPH781, Hubei Huangshi Medical Equipment Factory), refrigerator (Meiling), electric constant temperature water bath (DZKW-S-4, Beijing Yongguangming Medical Instrument Factory), quartz sand (analytical grade, Tianjin BASF Chemical Co., Ltd.), anhydrous ethanol (Tianjin Tianli Chemical Reagent Co., Ltd.)

[0061] 1.2 Stress treatment of soybean aphid by Orientia spp.

[0062] The soybean aphids of various ages were subjected to stress treatment with adult East Asian flower stink bugs. The treatment methods were divided into three types: contact stress, odor stress and visual stress. The control group was not subjected to East Asian flower stink bug stress. A certain number of soybean aphids were placed on the upper layer of each homemade culture dish, and the adult East Asian flower stink bugs were placed on the lower layer. The middle of the culture dish was isolated by a transparent visible plastic plate and an invisible air filter with pinholes. After treatment, it was placed in an incubator with a temperature of 24±1℃, a relative humidity of 70%±5%, and a light intensity of 16L:8D. After 24h, the stressed soybean aphids were taken for various biochemical substances.

[0063] 1.3 Enzyme solution preparation

[0064] Referring to the enzyme extraction method of Gao Huanhuan et al. (2016), after 24 hours, 5 instars of soybean aphids were selected in each treatment, and 5 surviving insects of each instar were collected and placed in a 1.5mL Eppendorf centrifuge tube. 400μL 0.02mol / L phosphate buffer (NaCl 136.89mM; KCl 2.67mM; Na2HPO4 8.1mM; KH2PO4 1.76mM) and a small amount of quartz sand were added, and the mixture was fully ground in an ice bath, and the ground homogenate was placed in a high-speed refrigerated centrifuge at 8000g and 4℃ for 10min. The supernatant was divided into a new 1.5mL Eppendorf centrifuge tube and placed on ice for testing. Three replicates were set for the 5 instars of each treatment.

[0065] 1.4 Determination of protein, reducing sugar and trehalose content

[0066] 1.4.1 Determination of protein content

[0067] The determination was carried out with reference to the Coomassie Brilliant Blue G-250 staining method of Bradford (1976). Coomassie Brilliant Blue G-250 solution and bovine serum albumin standard solution were prepared respectively to prepare a standard curve. 4 μL of enzyme solution was added to the sample well of the ELISA plate for each sample, 4 μL of phosphate buffer was added to the control well, and then 200 μL of rewarmed Coomassie Brilliant Blue G-250 reagent was added, mixed thoroughly, and placed at room temperature for 5 minutes. The ELISA reader was set at a wavelength of 595 nm for determination, and the OD value was recorded. Repeat 3 times, and the protein concentration in each sample to be tested was calculated according to the protein standard curve.

[0068] 1.4.2 Determination of reducing sugar and trehalose content

[0069] The content of trehalose was determined by the anthrone sulfuric acid method. Anthrone solution and 10 mg / mL trehalose standard solution were prepared to prepare a standard curve. For each sample, 60 μL of sample solution and 240 μL of prepared anthrone reagent were mixed in a 1.5 mL Eppendorf centrifuge tube, 95°C water bath for 10 min (cover tightly to prevent water loss), and naturally cooled to room temperature. 200 μL was taken to the ELISA plate, and the measurement was performed at a wavelength of 620 nm. The OD value was recorded and repeated 3 times. According to the trehalose standard curve, the trehalose content in each sample to be tested was calculated.

[0070] The content of reducing sugar was determined by DNS method. 3,5-dinitrosalicylic acid (DNS) alkaline solution and 10 mg / mL glucose standard solution were prepared to prepare standard curve. In the test tube, 175 μL sample solution and 125 μL prepared DNS reagent were mixed in a 1.5 mL Eppendorf centrifuge tube for each sample, heated in a boiling water bath for 5 min (covered tightly to prevent water loss), and immediately cooled to room temperature and mixed. In the control tube, 175 μL sample solution and 125 μL distilled water were mixed in a 1.5 mL Eppendorf centrifuge tube for each sample, heated in a boiling water bath for 5 min (covered tightly to prevent water loss), and immediately cooled to room temperature and mixed. Take 200 μL to a 96-well ELISA plate, measure at a wavelength of 540 nm, record the OD value, and repeat 3 times. The difference in absorbance between the test tube and the control tube was calculated, and the reducing sugar content in each sample was calculated based on the glucose standard curve (Wu Junxiang et al., 2004; Ding Huimei, 2011; Cheng Yuan et al., 2016).

[0071] 1.5 Determination of enzyme activity

[0072] The determination of the specific activity of AchE, CarE and GSTs enzymes was carried out according to the instructions in the kit of Beijing Solebow Technology Co., Ltd. The enzyme activity value was calculated according to the formula in the kit manual. The wavelengths for the determination of the specific activity of AChE, CarE and GSTs enzymes were 412, 450 and 340 nm, respectively. The AchE activity unit is defined as the production of 1 nmol of molar extinction coefficient (TNB) per minute per milligram of tissue protein as one unit of enzyme activity; the CarE activity unit is defined as the increase of 1 catalytic absorbance per minute per milliliter of reaction system per milligram of tissue protein at 37°C as one unit of enzyme activity; the GST activity unit is defined as the catalysis of 1 μmol of 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) per minute per milligram of protein at 25°C or 37°C as one unit of enzyme activity.

[0073] 1.6 Data Analysis

[0074] All experimental data were analyzed and plotted using Microsoft Excel and SPSS Base Ver.19.0, and Duncan's new multiple range test was used to analyze the differences among treatments. One-way ANOVA was used to analyze the protein, trehalose and reducing sugar contents and the activities of AchE, CarE and GSTs in soybean aphids after being stressed by Orientia spp.

[0075] 2 Results

[0076] 2.1 Effects of Ornithogalum spp. stress on protein content in soybean aphids

[0077] After 24 hours of stress from the Oriental flower bug to soybean aphids, the protein content of the enzyme solution showed an increasing trend as the age of the aphids increased. Figure 1 The results showed that compared with the control group, at the adult aphid stage, the protein content of enzyme solution in the odor stress and visual stress treatment groups decreased by 23.42% and 57.27%, respectively, and the difference between the visual stress treatment group and other groups was extremely significant, while there was no significant difference between the direct contact stress and the control group; at the 4th instar, compared with the control group, the protein content of the odor stress and visual stress treatment groups decreased by 46.36% and 61.85%, respectively, while the direct contact stress decreased by 44.33%; at the 3rd instar, the protein content of the odor stress and visual stress treatment groups decreased by 27.99% and 30.15%, respectively, while the direct contact stress decreased by 3. 1.54%, and there was no significant difference among the three treatment groups; at the 2nd age, the protein content of the odor stress and visual stress treatment groups decreased by 15.82% and 24.64%, respectively, and that of direct contact stress decreased by 52.73%; at the 1st age, the protein content of the odor stress, visual stress and direct contact stress treatment groups decreased by 15.13%, 33.32% and 57.01%, respectively, and the protein content in the direct contact stress treatment group reached the lowest value (9.12 μg / mL), which was significantly lower than that of the control group, odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0078] 2.2 Effects of Ornithogalum spp. stress on trehalose content in soybean aphids

[0079] After 24 hours of stress from the Oriental flower stink bug to soybean aphid, the trehalose content in the stink bug showed an increasing trend as the age increased. Figure 2 The results showed that compared with the control group, at the adult aphid stage, the trehalose content in the odor stress and visual stress treatment groups increased by 97.77% and 43.49%, respectively, and the direct contact stress treatment group reached the highest value (3.4607 mg / mgprot), and there were extremely significant differences between the three stress methods and the control group; at the 4th instar, the trehalose content in the odor stress and visual stress treatment groups increased by 67.08% and 32.56%, respectively, and the direct contact stress increased by 104.12%; at the 3rd instar, the odor stress treatment group increased by 104.12%, and the visual stress treatment group increased by 104.12%. The trehalose content of the odor stress and visual stress treatment groups increased by 52.29% and 22.22% respectively, and the direct contact stress increased by 115.33%; at the 2nd instar, the trehalose content of the odor stress and visual stress treatment groups increased by 72.21% and 30.22% respectively, and the direct contact stress increased by 145.48%; at the 1st instar, the odor stress, visual stress and direct contact stress treatment groups increased by 57.24%, 37.96% and 160.72% respectively, and there was no significant difference between the odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0080] 2.3 Effects of Ornithogalum spp. stress on reducing sugar content in soybean aphids

[0081] After 24 hours of stress from the Oriental flower stink bug to soybean aphid, the reducing sugar content of the aphid showed a decreasing trend as the age increased. Figure 3 The results showed that compared with the control group, at the adult aphid stage, there was no significant difference in reducing sugar content between the odor stress, visual stress and direct contact stress treatment groups and the control group; at the 4th instar, the reducing sugar content of the odor stress and visual stress treatment groups increased by 27.29% and 11.36%, respectively, and that of direct contact stress increased by 46.10%, and the reducing sugar content of the three treatment groups was significantly higher than that of the control group, while that of the direct contact stress treatment group was extremely significantly higher than that of the odor stress and visual stress treatment groups; at the 3rd instar, the reducing sugar content of the odor stress and visual stress treatment groups increased by 69.72% and 55.27%, respectively, and that of direct contact stress increased by 120%, and the odor stress treatment group increased by 120%. There was no significant difference between the stress and visual stress treatment groups; at the second age, the reducing sugar content of the odor stress and visual stress treatment groups increased by 122.67% and 114.88%, respectively, and that of direct contact stress increased by 168.73%; at the first age, the reducing sugar content of the odor stress, visual stress and direct contact stress treatment groups increased by 308.95%, 193.53% and 371.54%, respectively, and the reducing sugar content in the direct contact stress treatment group reached the highest value (2.89 μg / mgprot), which was significantly higher than that of the control group, odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0082] 2.4 Effects of Ornithogalum spp. stress on the specific activity of acetylcholinesterase in soybean aphids

[0083] After 24 hours of stress from the Oriental flower stink bug to soybean aphid, the acetylcholinesterase activity of the stink bug showed a decreasing trend as the age of the stink bug increased. Figure 4The results showed that compared with the control group, at the adult aphid stage, the acetylcholinesterase activity in the odor stress and visual stress treatment groups decreased by 68.34% and 70.57%, respectively, and there was no significant difference between the odor stress and visual stress treatment groups, while there was no significant difference between the direct contact stress and the control group; at the 4th instar, the acetylcholinesterase activity in the odor stress and direct contact stress treatment groups increased by 56.91% and 104.57%, respectively, and the odor stress and direct contact stress treatment groups were significantly higher than the control group, while there was no significant difference between the visual stress treatment group and the control group; at the 3rd instar, the odor stress and visual stress treatment groups increased by The acetylcholinesterase activity in the odor stress and visual stress treatment groups increased by 368.71% and 136.04%, respectively, while that in the direct contact stress treatment groups increased by 918.05%. At the age of 1, the acetylcholinesterase activity in the odor stress, visual stress and direct contact stress treatment groups increased by 4228.37%, 1628.48% and 8506.98%, respectively, and the acetylcholinesterase activity in the direct contact stress treatment group reached the highest value (198.79 U / mg prot), which was significantly higher than that in the control group, odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0084] 2.5 Effects of Ornithogalum spp. stress on the specific activity of carboxylesterase in soybean aphids

[0085] After 24 hours of stress on soybean aphids by the Oriental flower stink bug, the specific activity of carboxylesterase showed a significant downward trend with the increase of age. Figure 5The results showed that compared with the control group, at the adult aphid stage, the carboxylesterase activity of the odor stress and visual stress treatment groups increased by 174.30% and 887.64%, respectively, and the visual stress and odor stress treatment groups were significantly higher than the control group, while there was no significant difference between the direct contact stress and the control group; at the 4th instar, the carboxylesterase activity of the odor stress and visual stress treatment groups increased by 126.16% and 586.81%, respectively, and the direct contact stress increased by 124.52%, and the three stress methods were significantly higher than the control group, while there was no significant difference between the odor stress and direct contact stress treatment groups; at the 3rd instar, the odor stress and visual stress treatment groups were significantly higher than the control group, while there was no significant difference between the odor stress and direct contact stress treatment groups. The activity of carboxylesterase in the odor stress and visual stress treatment groups increased by 55.48% and 1508.95% respectively, while that in direct contact stress increased by 173.87%, and there were extremely significant differences among the three treatment groups; at the 2nd age, the activity of carboxylesterase in the odor stress and visual stress treatment groups increased by 1135.72% and 875.35% respectively, while that in direct contact stress increased by 581.85%; at the 1st age, the activity of carboxylesterase in the odor stress, visual stress and direct contact stress treatment groups increased by 1889.46%, 357.15% and 1713.34% respectively, and the activity of carboxylesterase in the odor stress treatment group reached the highest value (0.0038U / mg prot), which was significantly higher than that in the control group, odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0086] 2.6 Effects of Ornithogalum spp. stress on the specific activity of glutathione-S-transferase in soybean aphids

[0087] After 24 hours of stress on soybean aphids by the Oriental flower stink bug, the specific activity of glutathione-S-transferase showed a significant downward trend with the increase of age. Figure 6The results showed that compared with the control group, at the adult aphid stage, the glutathione-S-transferase activity in the odor stress and visual stress treatment groups increased by 97.62% and 229.91%, respectively, while that in the direct contact stress treatment group decreased by 58.5%. The differences between the three treatment groups and the control group were extremely significant, while the glutathione-S-transferase activity in the direct contact stress treatment group was extremely significantly lower than that in the control group. At the 4th instar, the glutathione-S-transferase activity in the odor stress and visual stress treatment groups increased by 81.78% and 219.26%, respectively, while that in the direct contact stress treatment group decreased by 12.19%. There was no significant difference between the stress treatment group and the control group; at the 3rd instar, the odor stress and visual stress treatment groups increased by 20.55% and 10%, respectively, while the direct contact stress decreased by 58.45%; at the 2nd instar, the glutathione-S-transferase specific activity of the odor stress and visual stress treatment groups increased by 31.17% and 7.39%, respectively, while the direct contact stress increased by 156.59%, and the direct contact stress and odor stress treatment groups were significantly higher than the control group, and the glutathione-S-transferase specific activity in the direct contact stress treatment group reached the highest value (0.06nmol / min / mg prot), while there was no significant difference between the visual stress treatment group and the control group; at 1 year old, the odor stress, visual stress and direct contact stress treatment groups increased by 10.23%, 2.54% and 104.42%, respectively, and the direct stress was significantly higher than the control group, odor stress and visual stress treatment groups (F=167.694, df=3, P<0.001).

[0088] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected, and the scope of the present invention to be protected is defined by the attached claims and their equivalents.

Claims

1. A method for verifying that the stress of Orius sauteri affects the activity of endogenous substances in soybean aphids, wherein the test insects are Orius sauteri and soybean aphid Aphis glycines, the Orius sauteri is collected from the soybean field of Xiangyang Experimental Farm in Heilongjiang Province, and is raised in a constant temperature incubator with a temperature of T=25±1°C, a relative humidity of RH=70%±5%, and a light intensity of L:D=16:8, and is fed with soybean aphids, and the Orius sauteri is propagated using soybean sprouts as ovipositors; the soybean aphid Aphis glycines is a local sensitive population of Harbin that has been retained in the laboratory for a long time, and is raised in artificial potted soybean seedlings, which are placed in an artificial climate box with a temperature of 25±1°C, a relative humidity of 65%-70%, and a photoperiod of 14L:10D, and the test soybean variety is Dongnong 52, characterized in that: The method for treating soybean aphids with stress of East Asian flower stink bugs is as follows: using adult East Asian flower stink bugs to treat soybean aphids of various ages with stress, and the treatment methods are divided into three types: contact stress, odor stress and visual stress, and a control group is subjected to no East Asian flower stink bug stress; a certain number of soybean aphids are placed in the upper layer of each homemade culture dish, and adult East Asian flower stink bugs are placed in the lower layer, and the middle of the culture dish is isolated by a transparent visible plastic plate and an invisible air permeable filter with pinholes; after the treatment, the dish is placed in a culture box with a temperature of 24±1°C, a relative humidity of 70%±5% and a light intensity of 16L:8D; after 24 hours, the stressed soybean aphids are taken to respectively carry out various biochemical substance determinations.

2. A method for verifying that the stress of Ornithogalum spp. affects the activity of endogenous substances in soybean aphids according to claim 1, characterized in that: The results of the effect of East Asian flower stink bug stress on the protein content of soybean aphids are as follows: 24 hours after the East Asian flower stink bug stressed soybean aphids, the protein content of the enzyme solution showed an increasing trend with the increase of the age; compared with the control group, at the adult aphid stage, the protein content of the enzyme solution in the odor stress and visual stress treatment groups decreased by 23.42% and 57.27%, respectively, and the difference between the visual stress treatment group and other groups was extremely significant, while there was no significant difference between the direct contact stress and the control group; at the 4th instar, compared with the control group, the protein content of the odor stress and visual stress treatment groups decreased by 46.36% and 61.85%, respectively, and the direct contact stress decreased by 44.33%; at the 3rd instar, the odor stress and visual stress treatment components At the age of 2, the protein contents of the odor stress and visual stress treatment groups decreased by 15.82% and 24.64%, respectively, while that of the direct contact stress treatment groups decreased by 52.73%. At the age of 1, the protein contents of the odor stress, visual stress and direct contact stress treatment groups decreased by 15.13%, 33.32% and 57.01%, respectively, and the protein content in the direct contact stress treatment group reached the lowest value: 9.12 μg / mL, which was significantly lower than that of the control group, odor stress and visual stress treatment groups: F=167.694, df=3, P<0.

001.

3. A method for verifying that the stress of Ornithogalum spp. affects the activity of endogenous substances in soybean aphids according to claim 1, characterized in that: The results of the effect of East Asian flower stink bug stress on the trehalose content of soybean aphids are as follows: after 24 hours of East Asian flower stink bug stress on soybean aphids, the trehalose content showed an increasing trend with the increase of age; compared with the control group, at the adult aphid stage, the trehalose content in the odor stress and visual stress treatment groups increased by 97.77% and 43.49%, respectively, and the direct contact stress treatment group reached the highest value: 3.4607mg / mg prot, while there were extremely significant differences between the three stress methods and the control group; at the 4th age, the trehalose content of the odor stress and visual stress treatment groups increased by 67.08% and 32.56%, respectively, and that of direct contact stress increased by 104.12%; at the 3rd age, the trehalose content of the odor stress and visual stress treatment groups increased by 52.29% and 22.22%, respectively, and that of direct contact stress increased by 115.33%; at the 2nd age, the trehalose content of the odor stress and visual stress treatment groups increased by 72.21% and 30.22%, respectively, and that of direct contact stress increased by 145.48%; at the 1st age, the trehalose content of the odor stress, visual stress and direct contact stress treatment groups increased by 57.24%, 37.96% and 160.72%, respectively, while there was no extremely significant difference between the odor stress and visual stress treatment groups: F=167.694, df=3, P<0.

001.

4. A method for verifying that the stress of Ornithogalum spp. affects the activity of endogenous substances in soybean aphids according to claim 1, characterized in that: The results of the effect of East Asian flower stink bug stress on the reducing sugar content of soybean aphids were as follows: after 24 hours of East Asian flower stink bug stress on soybean aphids, the reducing sugar content showed a decreasing trend with the increase of age; compared with the control group, at the adult aphid stage, there was no significant difference in reducing sugar content between the odor stress, visual stress and direct contact stress treatment groups and the control group; at the 4th instar, the reducing sugar content of the odor stress and visual stress treatment groups increased by 27.29% and 11.36%, respectively, and the direct contact stress increased by 46.10%, and the reducing sugar content of the three treatment groups was significantly higher than that of the control group, and the reducing sugar content of the direct contact stress treatment group was extremely significantly higher than that of the odor stress and visual stress treatment groups. ; At the 3rd instar, the reducing sugar contents in the odor stress and visual stress treatment groups increased by 69.72% and 55.27%, respectively, while that in the direct contact stress treatment group increased by 120%, and there was no significant difference between the odor stress and visual stress treatment groups; At the 2nd instar, the reducing sugar contents in the odor stress and visual stress treatment groups increased by 122.67% and 114.88%, respectively, while that in the direct contact stress treatment group increased by 168.73%; At the 1st instar, the reducing sugar contents in the odor stress, visual stress and direct contact stress treatment groups increased by 308.95%, 193.53% and 371.54%, respectively, and the reducing sugar content in the direct contact stress treatment group reached the highest value: 2.89μg / mg prot, which was significantly higher than that in the control group, odor stress and visual stress treatment groups: F=167.694, df=3, P<0.

001.

5. A method for verifying that the stress of Ornithogalum spp. affects the activity of endogenous substances in soybean aphids according to claim 1, characterized in that: The results of the effect of East Asian flower stink bug stress on the specific activity of acetylcholinesterase of soybean aphids were as follows: after 24 hours of stress by East Asian flower stink bugs on soybean aphids, the specific activity of acetylcholinesterase showed a decreasing trend with the increase of age; compared with the control group, at the adult aphid stage, the specific activity of acetylcholinesterase in the odor stress and visual stress treatment groups decreased by 68.34% and 70.57%, respectively, and there was no significant difference between the odor stress and visual stress treatment groups, while there was no significant difference between the direct contact stress and the control group; at the 4th instar, the specific activity of acetylcholinesterase in the odor stress and direct contact stress treatment groups increased by 56.91% and 104.57%, respectively, and the odor stress and direct contact stress treatment groups were significantly higher than the control group, while there was no significant difference between the visual stress treatment group and the control group; at the 3rd instar, the odor stress and visual stress treatment groups increased by 10. The acetylcholinesterase activity of the odor stress and visual stress treatment groups increased by 115.19% and 28.70% respectively, while that of the direct contact stress treatment groups increased by 174.31%. There were significant differences among the three treatment groups and they were all significantly higher than the control group. At the 2nd age, the acetylcholinesterase activity of the odor stress and visual stress treatment groups increased by 368.71% and 136.04% respectively, while that of the direct contact stress treatment groups increased by 918.05%. At the 1st age, the acetylcholinesterase activity of the odor stress, visual stress and direct contact stress treatment groups increased by 4228.37%, 1628.48% and 8506.98% respectively, and the acetylcholinesterase activity of the direct contact stress treatment group reached the highest value: 198.79 U / mgprot, which was significantly higher than that of the control group, odor stress and visual stress treatment groups: F=167.694, df=3, P<0.

001.

6. A method for verifying that the stress of Ornithogalum spp. affects the activity of endogenous substances in soybean aphids according to claim 1, characterized in that: The results of the effect of East Asian flower stink bug stress on the specific activity of carboxylesterase of soybean aphids are as follows: after 24 hours of stress by East Asian flower stink bugs on soybean aphids, the specific activity of carboxylesterase showed a significant downward trend with the increase of age; compared with the control group, at the adult aphid stage, the specific activity of carboxylesterase in the odor stress and visual stress treatment groups increased by 174.30% and 887.64%, respectively, and the visual stress and odor stress treatment groups were significantly higher than the control group, while there was no significant difference between the direct contact stress and the control group; at the 4th instar, the specific activity of carboxylesterase in the odor stress and visual stress treatment groups increased by 126.16% and 586.81%, respectively, and the direct contact stress increased by 124.52%, and the three stress methods were significantly higher than the control group, There was no significant difference between the odor stress and direct contact stress treatment groups; at the 3rd instar, the odor stress and visual stress treatment groups increased by 55.48% and 1508.95%, respectively, while the direct contact stress increased by 173.87%, and there were extremely significant differences among the three treatment groups; at the 2nd instar, the carboxylesterase specific activity of the odor stress and visual stress treatment groups increased by 1135.72% and 875.35%, respectively, while the direct contact stress increased by 581.85%; at the 1st instar, the odor stress, visual stress and direct contact stress treatment groups increased by 1889.46%, 357.15% and 1713.34%, respectively, and the carboxylesterase specific activity in the odor stress treatment group reached the highest value: 0.0038U / mg prot, which was significantly higher than that of the control group, odor stress and visual stress treatment groups: F=167.694, df=3, P<0.

001.

7. A method for verifying that the stress of Ornithogalum spp. affects the activity of endogenous substances in soybean aphids according to claim 1, characterized in that: The results of the effect of East Asian flower stink bug stress on the glutathione-S-transferase activity of soybean aphids were as follows: after 24 hours of stress by East Asian flower stink bugs on soybean aphids, the glutathione-S-transferase activity showed a significant downward trend with the increase of age; compared with the control group, at the adult aphid stage, the glutathione-S-transferase activity in the odor stress and visual stress treatment groups increased by 97.62% and 229.91%, respectively, while the direct contact stress decreased by 58.5%, and the differences between the three treatment groups and the control group were extremely significant, while the glutathione-S-transferase activity in the direct contact stress treatment group was extremely significantly lower than that in the control group; at the 4th instar, the glutathione-S-transferase activity in the odor stress and visual stress treatment groups increased by 8 At the age of 3, the glutathione-S-transferase activity in the odor stress and visual stress treatment groups increased by 20.55% and 10%, respectively, while that in the direct contact stress treatment group decreased by 58.45%. At the age of 2, the glutathione-S-transferase activity in the odor stress and visual stress treatment groups increased by 31.17% and 7.39%, respectively, while that in the direct contact stress treatment group increased by 156.59%. Both the direct contact stress and odor stress treatment groups were significantly higher than the control group. The glutathione-S-transferase activity in the direct contact stress treatment group reached the highest value: 0.06nmol / min / mg prot, while there was no significant difference between the visual stress treatment group and the control group; at 1 year old, the odor stress, visual stress and direct contact stress treatment groups increased by 10.23%, 2.54% and 104.42%, respectively, and the direct stress was significantly higher than the control group, odor stress and visual stress treatment groups: F=167.694, df=3, P<0.001.

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