Method for improving host searching capability of beneficial bugs by utilizing olfaction training

Abstract

The invention discloses a method for improving host searching ability of beneficial bugs by utilizing olfaction training, which comprises the following steps: setting four treatments, namely a hunger group, a food stimulation group, a smell stimulation group and a training group, for both male and female adults, setting the hunger group in order to display the training effect, and setting the food stimulation group and the smell stimulation group in order to improve the host searching ability of the beneficial bugs. The food stimulation group and the smell stimulation group are treated as a blank test. The method comprises the following steps: comparing a test of taking only food as non-conditional stimulation with a conditional stimulation test of staggering conditional stimulation and non-conditional stimulation, and performing relevance learning on the beneficial bugs by giving relevance alternative food and smell stimulation to insects, thereby improving the host searching capability of the beneficial bugs and improving the searching efficiency of the beneficial bugs. And identification and preference of the beneficial bugs to plant varieties can be realized through training.

Description

Technical Field

[0001] The invention relates to the technical field of natural enemy insect predation training, and in particular to a method for improving the host search ability of beneficial bugs by utilizing olfactory training, which is used for performing olfactory training on the beneficial bugs to improve their predation efficiency. Background Art

[0002] Picromerus lewisi (Scott) belongs to the order Hemiptera, family Pentatomidae, subfamily Asopinae. It is widely distributed in various provinces and cities in my country and in Asian regions such as Japan and North Korea. Its nymphs and adults can prey on the larvae of various lepidopteran pests such as S. litura, M. separata and H. assulta, and is a very potential predatory natural enemy in agriculture. However, due to the phenomenon of deterioration of predation ability caused by the decline of host location ability of large-scale artificially raised natural enemies, combined with the current practice of using beneficial bugs, after releasing beneficial bugs in the field, due to the strong migration and diffusion ability of beneficial bugs, the number of them in the target field fluctuates greatly, which has a certain adverse effect on long-term field pest control. In addition, natural enemies have no preference for plant varieties after being released in the wild, resulting in random spread in the wild, which poses certain risks to the ecosystem and affects farmers' enthusiasm for using natural enemies to control pests. When natural enemies prey on pests, they rely on pest-inducing volatiles released by plants when the pests harm plants to locate the host, thereby searching for pests at a long distance and completing the predation process. Using olfactory learning to regulate insect directional behavior is a suitable method to improve the natural enemy's ability to locate the host, thereby improving the natural enemy's ability to control pests. Olfactory learning is commonly studied in Diptera and Hymenoptera insects, mainly for pollinating insects and natural enemies in Diptera and Hymenoptera to improve pollination and study the mechanism of harming plants. At present, the main research method for olfactory learning of natural enemy insects is to expose the natural enemy insects to a certain odor environment containing host chemical information. The parasitic or feeding experience in this odor environment enhances the natural enemy insect's preference for the host. However, when the above method is used to train the natural enemy insects for olfaction, the natural enemy insects are free to feed and move in the odor environment, and the artificial precise control of the order, time and frequency of conditioned stimuli and unconditioned stimuli is not achieved. Therefore, it is difficult to explain the specific conditions for the generation of this learning behavior. In addition, this training method is time-consuming and is not suitable for multiple training in a short period of time to consolidate the learning effect. Classical conditioning can achieve precise control over the duration and frequency of conditioned stimuli (CS) and unconditioned stimuli (US), and is therefore widely used in studies of associative olfactory learning in insects. However, it has not yet been applied to olfactory learning of natural enemy insects. Summary of the invention

[0003] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for improving the host-searching ability of beneficial bugs by utilizing olfactory training, utilizing the pest-inducing volatiles released by tobacco plants after the larvae of the main tobacco pest Spodoptera litura damage tobacco as conditioned stimuli, and using the food sticky insects in the beneficial bug breeding process as unconditioned stimuli. By training by controlling the conditioned and unconditioned stimuli, a beneficial bug population with a stronger host-searching ability can be achieved, and a beneficial bug population with a certain preference for different varieties of host plants can be cultivated.

[0004] In order to achieve the above object, the technical scheme of the present invention is as follows: a method for improving the host search ability of beneficial bugs by using olfactory training, comprising the following steps:

[0005] The adult beneficial bugs within 24 hours of emergence were picked out from the insect cage, placed in a clean insect box, sealed with medical gauze and rubber bands, starved for 48 hours before the test, without food and water, and the male and female adults were tested separately;

[0006] Place 10 beetles in a plastic box in advance and let them adapt for 10 minutes. Wrap the roots and soil of the tobacco plants used in the experiment with tin foil and place them in a beetle training device with good airtightness.

[0007] Four treatments were set for both male and female adults, namely, starvation group, food stimulation group, odor stimulation group, and training group;

[0008] The starvation group received no treatment after 48 h of starvation;

[0009] The food stimulation group was fed with sticky insects three times after 48 h of starvation, with each feeding lasting 5 min and an interval of 40 min between each feeding;

[0010] After starvation for 48 hours, the odor stimulation group was fed with sticky insects three times, each feeding lasting 5 minutes, with an interval of 40 minutes between each feeding. Six hours after the feeding, tobacco pest-inducing volatiles were introduced to the group for stimulation three times, each lasting 4 minutes, with an interval of 40 minutes between each stimulation.

[0011] After starvation for 48 hours, the training group was first stimulated with tobacco pest-induced volatiles for 1 minute, and then fed with sticky insects. The tobacco pest-induced volatiles were continued for 3 minutes, and then the volatiles were removed. The mice were allowed to continue feeding for 5 minutes, and then the sticky insects were removed. This process was one training session, and the training was conducted 3 times, with an interval of 40 minutes between each training session.

[0012] After the experiment, the beneficial bugs were taken out of the plastic box and placed in a clean insect breeding box without food or water. The box was sealed with medical gauze and rubber bands to prevent escape.

[0013] Optionally, the beneficial stink bug training device includes a glass dryer, in which the roots and soil of tobacco plants are wrapped with tin foil and placed in the glass dryer; the glass dryer is provided with an air outlet and an air inlet, and the air inlet is connected to the air outlet of an air pump and two closed containers respectively filled with activated carbon and pure water by a glass conduit and a silicone tube in sequence, and the air outlet is connected to a glass rotor flowmeter, and during training, the air outlet of the flowmeter directly leads to the container containing the beneficial stink bugs.

[0014] Furthermore, the flow rate of the flow meter was adjusted to 400 ml / min, and the test was started after the flow rate was stabilized.

[0015] Optionally, after use, the plastic box is cleaned three times with water, 95% alcohol and water in sequence, and then dried for later use.

[0016] Preferably, during the training process of the training group, tobacco pest-induced volatiles are used as conditioned stimuli and food is used as unconditioned stimuli, and the two overlap in time.

[0017] Furthermore, after the training of the beneficial bugs is completed, a learning effect verification is performed: the beneficial bugs with poor vitality are removed from the hunger group, food stimulation group, odor stimulation group and training group, and the food stimulation group, odor stimulation group and training group are starved for 24 hours before the learning effect verification is performed, and the hunger group does not need to be starved again.

[0018] Due to the adoption of the above technical scheme, the advantages of the present invention are: the present invention uses the classical conditioned reflex method composed of conditioned stimulus and unconditioned stimulus for the first time to realize the olfactory training of predatory natural enemy insects, and solves the current problem of decreased predation efficiency after large-scale artificial breeding of natural enemy insects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0020] Figure 1 This is a connection diagram of the beneficial bug training device.

[0021] Figure 2 This is a schematic diagram of the olfactory training method for beneficial bugs;

[0022] Note: Starvation: starvation treatment; US: food stimulation (stick insects); CS: odor stimulation (tobacco insect pest-induced volatiles); US and CS: food stimulation and odor stimulation were performed simultaneously.

[0023] Figure 3 This is a schematic diagram of the connection of the Y-type olfactometer.

[0024] Figure 4 This is the result of Y-tube selection; the chi-square test was used to analyze the selection bias, n>40.

[0025] In the figure: A. The host preference test results of female beneficial bugs in different treatment groups using tobacco variety Yunyan 87, B. The host preference test results of male beneficial bugs in different treatment groups using tobacco variety Yunyan 87, C. The host preference test results of female beneficial bugs in different treatment groups using tobacco variety Yunyan 116, D. The host preference test results of male beneficial bugs in different treatment groups using tobacco variety Yunyan 116.

[0026] Figure 5 is the selection rate of Y-type tube; Pearson chi-square was used to compare the differences in selection rates among the groups, * indicates that the difference is statistically significant at the p < 0.05 level, n > 40.

[0027] Figure 6 is the time when the beneficial bugs arrive at the tobacco plants; One-way Anova LSD method was used to compare the differences among the groups, error bars represent standard error of the mean (SEM), n≥30, ** and *** indicate statistically significant differences at p<0.01 and p<0.001, respectively.

[0028] Figure 7 The variety preference of the beneficial bug for Yunyan 87 and Yunyan 116. The chi-square test was used to analyze the variety preference, n≥30. DETAILED DESCRIPTION

[0029] The principles and features of the present invention are described below in conjunction with the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0030] 1. Material selection: Two main tobacco varieties, Yunyan 87 and Yunyan 116, were selected for the experiment. Tobacco was planted in an artificial simulated climate chamber with a temperature of 25℃-28℃, a humidity of 60%-70%, and a photoperiod of 14L:10D. After sowing in a seedling tray (hole depth 5cm, upper diameter 4cm, lower diameter 2cm) for 30 days, it was transplanted into a double-color pot (upper diameter 14cm, lower diameter 12cm, height 13cm), and continued to grow for 30 days. Tobacco with good growth and no pests and diseases was selected for the experiment. The nutrient soil for planting tobacco was provided by the Wuligang Seedling Base in Yuzhang Town, Xingren City and the Tobacco Leaf Workstation in Yuzhang Town. Spodoptera litura was purchased from Henan Keyun Biotechnology Co., Ltd. and raised in an artificial simulated climate chamber with a temperature of 25±3℃ and a relative humidity of 35%-45% (newly hatched larvae require a relative humidity of 60%-70%). It was raised under shaded conditions, with no additional light except natural light, and fed with fresh tobacco leaves. The beneficial bugs and armyworms were provided by the beneficial bug breeding base in Qianxinan Prefecture and were raised in an artificial simulated climate chamber. Armyworms were raised under shaded conditions, with no additional light except natural light, at a temperature of 25±3℃, and young larvae were fed with fresh young corn leaves, with a relative humidity of 60%-70%. After the third instar, they were fed with artificial feed and fresh young corn leaves, with a relative humidity of 35%-45%. The beneficial bugs were raised under 24-hour light conditions, at a temperature of 25±3℃, and a relative humidity of 60%-70%. Newly hatched larvae were fed with 10% honey water, and after the second instar, they were fed with armyworms.

[0031] 2. Treatment of tobacco plants before the test: 6 early 4th-instar Spodoptera litura that had been starved for 6 hours were placed on the 3rd and 4th leaves from top to bottom of the tobacco plants, and the tobacco plants were placed in insect cages to prevent the Spodoptera litura from escaping. When the Spodoptera litura had fed on more than 20% of the leaf area, the Spodoptera litura was removed, and the test was carried out between 18h and 30h after the Spodoptera litura began to feed.

[0032] Embodiment 1:

[0033] A method for improving the host search ability of beneficial bugs by using olfactory training, wherein the training group is a method of training by classical conditioning, and at the same time, in order to demonstrate the training effect, three groups of treatments are set up, namely, a hunger group, a food stimulation group and an odor stimulation group as a blank test, an unconditioned stimulus test with only food as the unconditioned stimulus, and a conditioned stimulus experiment in which the conditioned stimulus and the unconditioned stimulus are staggered. In addition, because there are obvious differences between male and female insects in achieving host location using volatiles, the present invention separates male and female insects to compare the difference in training effects between male and female beneficial bugs. Specifically, the following steps are included:

[0034] The beneficial bug adults within 24 hours of emergence were picked out from the insect cage and placed in a clean insect box (19.5 cm × 13.4 cm × 7.3 cm). The box was sealed with medical gauze and rubber bands. They were starved for 48 hours before the test without food and water. Male and female adults were tested separately.

[0035] Place 10 beneficial bugs in a plastic box (6 cm in diameter, 3 cm in height) in advance and let them adapt for 10 minutes. Wrap the roots and soil of the tobacco plants with tin foil and place them in the beneficial bug training device, such as Figure 1 As shown, the beneficial stink bug training device includes a glass dryer with good airtightness, that is, the root system and soil of the tobacco plant are wrapped with tinfoil and placed in the glass dryer, the glass dryer is provided with an air outlet and an air inlet, and the air outlet of the air pump and two closed containers respectively equipped with activated carbon and pure water are connected with a glass conduit and a silicone tube before the air inlet, and the air outlet is connected to a glass rotor flowmeter (air 0.1-1L / min), and the flow meter air outlet directly leads to the container equipped with beneficial stink bugs during training. After the device is connected, the flow rate of the flowmeter is adjusted to 400ml / min, and the test is started after the flow rate stabilizes.

[0036] like Figure 2 As shown, four treatments were set for both male and female adults, namely, starvation group, food stimulation group, odor stimulation group and training group.

[0037] The starvation group received no treatment after 48 h of starvation.

[0038] The food stimulation group was fed with sticky insects three times after starvation for 48 h, with each feeding lasting 5 min and an interval of 40 min between each feeding.

[0039] After being starved for 48 hours, the odor stimulation group was first fed with sticky insects three times, with each feeding lasting 5 minutes and an interval of 40 minutes between each feeding. Six hours after the feeding, tobacco pest-inducing volatiles were introduced to them for stimulation three times, with each stimulation lasting 4 minutes and an interval of 40 minutes between each stimulation.

[0040] After starvation for 48 hours, the training group was first stimulated with tobacco pest-induced volatiles for 1 minute, and then fed with sticky insects, and continued to pass tobacco pest-induced volatiles for 3 minutes before removing the volatiles, allowing them to continue feeding for 5 minutes before removing the sticky insects. This process was one training session, and the training was conducted 3 times, with an interval of 40 minutes between each training session. That is, during the training process of the training group, tobacco pest-induced volatiles were used as conditioned stimuli and food was used as unconditioned stimuli. The two overlapped in time and were not stimulated separately.

[0041] After the experiment, the beneficial bugs were taken out of the plastic box and placed in a clean insect box (19.5 cm × 13.4 cm × 7.3 cm). They were not given food or water and the box was sealed with medical gauze and rubber bands to prevent escape.

[0042] After use, clean the plastic box with water, 95% alcohol and water three times in turn, dry it and set it aside.

[0043] The hungry group, food stimulation group, odor stimulation group and training group all removed the less active beneficial bugs, and the food stimulation group, odor stimulation group and training group were starved for 24 hours before verifying the learning effect. The hungry group did not need to be starved again.

[0044] Furthermore, after the training of the beetle is completed, the learning effect is verified, including:

[0045] 1. Y-type olfactometer test

[0046] like Figure 3 As shown, a tobacco plant infested by Spodoptera litura and a tobacco plant without any treatment were wrapped with tin foil, and the roots and soil were placed in two identical clean glass desiccators. The air outlet of the air pump and two closed containers containing activated carbon and purified water were connected in sequence before the air inlet of the glass desiccator. The two air outlets were connected to two glass rotor flowmeters (air 0.1-1L / min, Changzhou Shuanghuan Thermal Instrument Co., Ltd.) and two arms of the Y-type tube, and the Y-type tube was placed in a dark box, and a monochromatic light source (yellow light) was connected to the dark box to ensure that the light of the two arms of the Y-type tube was uniform. The angle between the two arms of the Y-type tube was 60°, the two arms were 10 cm long, the main arm was 18 cm long, the inner diameter was 15 mm, and the outer diameter was 20 mm. After the Y-type olfactometer was connected, the flow rate of the flowmeter was adjusted to 400 ml / min, and the test was started after the flow rate stabilized.

[0047] Before making a choice, the insect was first placed in a glass tube with an inner diameter equivalent to the wall of the Y-tube to adapt for 3-5 minutes, and then the outlet of the glass tube was opened, placed in a dark box, connected to the main arm of the Y-tube, and allowed to crawl into the Y-tube by itself. The timing started from the moment the insect crawled into the arm of the Y-tube. When it crossed half of the side arm of the Y-tube, it was considered a successful choice, the timing was stopped, and the direction of the choice was recorded. If it did not make a successful choice within 5 minutes, it was considered not to be a choice. Release one insect each time, swap the left and right positions of the two arms of the Y-tube after releasing three insects, and replace the Y-tube after releasing six insects. Clean it with water, 95% alcohol, and water in turn, and then dry it with an electric fan for standby use.

[0048] 2. Y-type olfactometer test results

[0049] The results of the Y-tube selection showed that the male and female adults of the starvation group, food stimulation group, odor stimulation group and training group all had a clear preference for tobacco plants infested by Spodoptera litura, and the difference in the training group was significantly higher (p < 0.001) than that in the other three treatments. Figure 4 shown.

[0050] The selection rate of beneficial bugs on tobacco plants infested by Spodoptera litura was calculated based on the selection results of the Y-shaped tube, and the differences in selection rates among the groups were compared using Pearson chi-square. The selection rates of male and female adults of beneficial bugs on tobacco plants in the training group were significantly higher than those in the other three treatments (p < 0.05). Figure 5 shown.

[0051] 1. Semi-field release experiment

[0052] In an open-air environment, a tobacco plant infested by Spodoptera litura was placed in a nylon mesh cage (120 mesh, 160cm×78cm×78cm). Ten beneficial bugs were placed in a plastic box (6cm in diameter, 3cm in height) in a group and adapted for 10 minutes in advance. The plastic box containing the beneficial bugs was placed in a nylon mesh cage, 1 meter away from the tobacco plant, the lid was opened, and the beneficial bugs were allowed to crawl out by themselves, and the timing was started. When the beneficial bugs arrived at the tobacco plant, the timing was stopped, and the time when the beneficial bugs arrived at the tobacco was recorded. If they still did not arrive after 30 minutes, it was considered as not arriving. Since beneficial bugs are more active in the evening, the test was fixed between 6pm and 8pm. During the test, the ambient temperature was above 20℃, and the tobacco plants were kept upwind of the beneficial bug release point.

[0053] Afterwards, the test of the variety preference of beneficial bugs for Yunyan 87 and Yunyan 116 was also conducted under the same conditions. One tobacco plant Yunyan 87 and one Yunyan 116 infested with Spodoptera litura were placed in a nylon mesh cage, and the beneficial bugs that had been trained with the pest-inducing volatiles of Yunyan 87 and received odor stimulation were released 1 meter away from the tobacco plants. The tobacco variety chosen by the beneficial bugs was recorded, and the left and right positions of two tobacco plants were exchanged once every 10 beneficial bugs were released.

[0054] 2. Results of the semi-field release experiment

[0055] Compared with the other three treatments, the time to reach the tobacco plants in the training group was significantly shortened. Figure 6 shown.

[0056] like Figure 7 As shown, male and female adults of beneficial bugs that were stimulated by the pest-inducing volatiles of Yunyan 87 showed no preference for Yunyan 87, but female beneficial bugs that were trained with the pest-inducing volatiles of Yunyan 87 showed a significant preference for Yunyan 87 (p=0.034), indicating that female beneficial bugs may have a certain recognition ability for the tobacco varieties they have been trained on.

[0057] In summary, training can achieve a strong host search ability. The results of the Y-type olfactometer test showed that the selection rate of male and female adults of the olfactory-trained beneficial bugs on tobacco plants infested by Spodoptera litura reached more than 80%, which was about 20% higher than that of the untrained beneficial bugs; in an open-air environment, under shrouded conditions, the beneficial bugs were released 1 meter away from the pest tobacco plants. The average time for the male and female adults who had been trained to reach the tobacco plants was shortened by about 50% compared with the untrained beneficial bugs, and the female beneficial bugs had a significant preference for the tobacco varieties that had been trained. And it is possible to carry out beneficial bug populations that have a certain preference for different varieties of host plants. It proves the theoretical feasibility of improving the predation efficiency of beneficial bugs on Spodoptera litura through domestication and creating beneficial bug behavior regulators, which lays the foundation for the subsequent development and application of large-scale domestication technology of beneficial bugs.

[0058] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the above specific implementation methods. Therefore, any modification or equivalent replacement of the present invention; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the invention are included in the scope of the claims of the present invention.

Claims

1. A method for improving the host-searching ability of beneficial bugs by using olfactory training, characterized in that: The following steps are involved: The adult beneficial bugs within 24 hours of emergence were picked out from the insect cage, placed in a clean insect box, sealed with medical gauze and rubber bands, starved for 48 hours before the test, without food and water, and the male and female adults were tested separately; Every 10 beneficial bugs were placed in a plastic box in advance to adapt for 10 minutes. The roots and soil of the experimental tobacco plants were wrapped with tin foil and placed in a beneficial bug training device with good airtightness. Four treatments were set for both male and female adults, namely, starvation group, food stimulation group, odor stimulation group, and training group; The starvation group received no treatment after 48 h of starvation; The food stimulation group was fed with sticky insects three times after 48 h of starvation, with each feeding lasting 5 min and an interval of 40 min between each feeding; After starvation for 48 hours, the odor stimulation group was fed with sticky insects three times, each feeding lasting 5 minutes, with an interval of 40 minutes between each feeding. Six hours after the feeding, tobacco pest-inducing volatiles were introduced to the group for stimulation three times, each lasting 4 minutes, with an interval of 40 minutes between each stimulation. After starvation for 48 hours, the training group was first stimulated with tobacco pest-induced volatiles for 1 minute, and then fed with sticky insects. The tobacco pest-induced volatiles were continued for 3 minutes, and then the volatiles were removed. The mice were allowed to continue feeding for 5 minutes, and then the sticky insects were removed. This process was one training session, and the training was conducted 3 times, with an interval of 40 minutes between each training session. After the experiment, the beneficial bugs were taken out of the plastic box and placed in a clean insect breeding box without food or water. The box was sealed with medical gauze and rubber bands to prevent escape.

2. The method for improving the host-seeking ability of beneficial bugs by using olfactory training according to claim 1, characterized in that: The beneficial stink bug training device comprises a glass dryer, in which the roots and soil of tobacco plants are wrapped with tin foil and placed; the glass dryer is provided with an air outlet and an air inlet, and the air inlet is connected to the air outlet of an air pump and two closed containers respectively filled with activated carbon and pure water by a glass conduit and a silicone tube in sequence, and the air outlet is connected to a glass rotor flowmeter, and during training, the air outlet of the flowmeter directly leads to the container filled with the beneficial stink bugs.

3. The method for improving the host-seeking ability of beneficial bugs by olfactory training according to claim 2, characterized in that: The flow rate of the flow meter was adjusted to 400 ml / min, and the test was started after the flow rate stabilized.

4. The method for improving the host-seeking ability of beneficial bugs by using olfactory training according to claim 1, characterized in that: After use, the plastic box is cleaned with water, 95% alcohol and water three times in sequence, and then dried for later use.

5. The method for improving the host-seeking ability of beneficial bugs by using olfactory training according to claim 1, characterized in that: In the training process of the training group, tobacco pest-induced volatiles are used as conditioned stimuli and food is used as unconditioned stimuli, and the two overlap in time.

6. The method for improving the host-seeking ability of beneficial bugs by using olfactory training according to claim 1, characterized in that: After the training of the beneficial bugs, the learning effect is verified: the beneficial bugs with poor vitality are removed from the hunger group, food stimulation group, odor stimulation group and training group, and the food stimulation group, odor stimulation group and training group are starved for 24 hours before the learning effect is verified. The hunger group does not need to be starved again.

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