An experimental device and method for detecting insect learning and memory

Abstract

The application belongs to the technical field of insect behavior science, and relates to an experimental device and method for detecting insect learning and memory. The device comprises an odor delivery device and a conditioning training device, and the application simultaneously creates a training paradigm of olfactory conditioned proboscis extension reflex based on volatile compounds as appetite stimulation. Through the device and the established training method, whether insects can establish the combined learning and memory ability of conditioned odor stimulation (CS) and volatile compound intake (US) is evaluated for the first time. The device can also evaluate the combined learning and memory ability of conventional classical odor stimulation (CS) and sugar food (non-volatile US) of insects. The application provides a device and method for evaluating the combined learning and memory ability of odor stimulation (CS) and volatile compound intake (US) of insects for the first time for global scientists and technical personnel, thereby leading and opening up a new research direction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of insect behavior science, and relates to an experimental device and method for detecting insect learning and memory, in particular to a device and method for detecting insect learning and memory driven by volatile chemicals. BACKGROUND

[0002] Olfactory conditioning of the proboscis extension reflex (Olfactory conditioning of PER) is a form of associative learning and memory. The establishment of this conditioned reflex requires the integration of two different types of external sensory information in the brain, namely olfactory information of odor, taste and other brain effects brought by the intake of chemical substances. Since Kimihisa Takeda (1961) successfully developed an insect olfactory conditioning proboscis extension reflex device and method, only sucrose food can be used as a non-conditional stimulus (US) paired with a certain conditional stimulus (CS, odor) for conditioning training to evaluate the food target-driven olfactory learning and memory ability of insects. This classic method has been widely used in many insect groups such as fruit flies and bees. For example, in 2019, Jinxi used the classical olfactory conditioning training method to conduct joint learning training of odor and food on male adults of Bactrocera dorsalis in the laboratory, i.e. 10% sucrose solution (non-volatile food, US) and sweet orange oil (volatile odor, CS) combined reward training, and used the proboscis extension reflex behavior as the criterion for determining whether learning or not, and measured the selection behavior and tropism behavior of Bactrocera dorsalis after learning. In other words, the existing domestic and foreign technology can only evaluate the association between non-volatile food (US) and volatile odor stimulus (CS), and thus evaluate the non-volatile substance-driven olfactory learning and memory ability of insects. However, so far, people have ignored a basic fact that in nature, there are a large number of volatile chemical substances (or food or drugs) that not only release odor for insect olfactory perception, but also can be actively ingested by insects to improve their survival and reproduction ability. For example, volatile aliphatic compounds, phenylpropanoid derivatives, monoterpenes, sesquiterpenes and their oxygen derivatives, volatile fatty acids in plant roots, stems, leaves, flowers, fruits and peels. However, it is not clear whether insects can establish the association between volatile US and volatile CS in the insect brain. These ingested chemical substances can also serve as odor substances due to their volatility. Therefore, using the existing device technology, the insect antennal olfactory system almost simultaneously receives the odor of the conditional odor stimulus (CS, such as ethyl acetate) and the non-conditional appetite stimulus substance, and the insect cannot determine which odor is the conditional stimulus, so the insect cannot establish the classical CS-US associative learning and memory. In other words, the volatility of the non-conditional appetite stimulus substance hinders the establishment of the conditional odor stimulus CS in the insect brain in our experiment. So far, people cannot evaluate the volatile appetite stimulus US-driven olfactory learning and memory ability of insects, and people can only stay at the level of traditional classical CS-US (sucrose) olfactory learning and memory research.

[0003] Therefore, it is necessary to develop a new insect conditioned proboscis extension reflex device and method, which can theoretically associate any odor (CS) with any volatile appetizing substance (US, such as methyl eugenol) for testing, thereby more deeply evaluating the learning and memory ability and survival ability of insects, serving the protection of insect biodiversity and human well-being. SUMMARY

[0004] In view of the above technical problems, the present application provides a device and method for detecting insect learning and memory (insect conditioned proboscis extension reflex), and the technical scheme is as follows:

[0005] An experimental device for detecting insect learning and memory, the device comprising a box made of opaque material, a pull-out door is arranged on the front of the box, and a smell delivery device and a conditioning training device are arranged in the box; the smell delivery device is a gas pump connected by a hose outside the box, and the gas pump is connected by a hose in sequence to an air filter, an integrated air humidifier / flow meter, a smell source chamber, and a smell release pipe; the conditioning training device is composed of an isolation chamber and an insect body fixator, the isolation chamber has a hexahedral structure with an open right face as an air outlet, the smell release pipe penetrates through the left side face of the isolation chamber and forms an angle of 15-45 degrees with the bottom face of the isolation chamber, and the isolation chamber must be kept horizontal to facilitate fine operation of the insect antennae, the left, front, rear and upper faces of the isolation chamber are composed of light transparent thin plates, the bottom face is wrapped with a film, a micropore is arranged in the center of the bottom film, the insect body fixator is located below the micropore of the isolation chamber, and an exhaust fan is arranged on the right side wall of the box.

[0006] Further, the insect body fixator is a fixed small tube with a backrest, and a hole support is used to stabilize the fixed small tube.

[0007] Further, the smell release pipe is a hard pipe with a diameter of ≤10 mm.

[0008] Further, a white light and an infrared light are arranged on the top of the box.

[0009] Further, the size of the box is 60*40*40 cm, and the size of the isolation chamber is 40*30*30 mm.

[0010] Further, the top of the box is provided with a adjustable fixing frame with a clamp for fixing the smell release pipe.

[0011] Preferably, a universal exhaust cover is arranged outside the box to avoid the accumulation of odors in the room.

[0012] The present application also provides a method for detecting insect learning and memory by using the device, comprising the following steps:

[0013] S1 Adaptation stage The day before the experiment, male D. citri with good activity and similar body size were selected in the rearing cage and placed in a 50-ml centrifuge tube with water for 12 h of starvation. On the day of the experiment, the insects were placed in an insect body fixer, and the tiny antennae were carefully introduced into the micro-hole at the bottom of the isolation chamber, with the antennae vertically upward, extending, and the gap was sealed with high-vacuum silicone grease. After 1 h of adaptation, the forelegs of the insects in the device were gently touched with pure water-soaked absorbent cotton to observe whether the insects exhibited a proboscis extension reflex. Insects that still exhibited a proboscis extension reflex after touching with pure water were removed, and the remaining insects were used in subsequent experiments. All conditioning training processes were performed under infrared light. The source substance of the conditioned stimulus odor was placed on filter paper (4 x 1 cm) in the odor source chamber. The odor in the filter paper in the odor source chamber was blown toward the antennae of the D. citri by air flow (1 L / min), and at the same time, multiple exhaust fans were opened on the side wall of the chamber to immediately blow away the various odors from the body of the insects, ensuring the success of the experiment. After each training, the filter paper was replaced.

[0014] S2 Conditioning training Before the start of the first training, the air flow was adapted for 15 s to reduce the disturbance caused by the change in the new environment and to reduce the stress of the insects. After the adaptation, the odor was blown toward the antennae of the male D. citri at a flow rate of 1 L / min. In the first training, the D. citri that exhibited a proboscis extension reflex to the odor were removed. After the release of the source substance of the conditioned stimulus odor for 3 s, the D. citri were given a solution of an appetite stimulant as a reward. The solution of the appetite stimulant was soaked in soft absorbent cotton, which was gently touched with the proboscis of the D. citri for 3 s. The release of the odor was maintained for 6 s. In the training of the CS-US pairing group, the D. citri underwent a total of 5 conditioning trainings, with an interval of 10 min between each training. The proboscis extension reflex to the source substance of the odor was recorded in each experiment. The solution of the appetite stimulant included volatile food and non-volatile food (e.g., sucrose).

[0015] S3 Memory extraction After the end of the training, the D. citri were taken out of the isolation chamber and placed in a room under an infrared lamp, maintaining a temperature of 25-26°C and a relative humidity of 50-60%. The D. citri were fed with a small amount of sucrose solution every 1 h to prevent excessive starvation and death. Then, memory extraction experiments were performed at 10 min, 24 h, and 48 h, respectively. D. citri that exhibited a proboscis extension reflex within 3 s of odor presentation were considered to have remembered the relationship between the conditioned stimulus (CS: source substance of the odor) and the unconditioned appetite stimulus, i.e., the CS became a signal predicting the reward, and were recorded as "learned". Other D. citri were recorded as "not learned".

[0016] S4 Statistical analysis uses dichotomous statistics to analyze the data of the olfactory conditioned proboscis extension reflex experiment (correct PER: 1, wrong or no PER: 0); the raw data is entered into Excel, the data is analyzed using IBM SPSS Statistics 26, and OriginPro 2021 is used for plotting; the performance changes caused by the number of training times within a single learning curve are detected using Cochran Q; the comparison between groups of learning curves uses repeated measures ANOVA; the inter-group difference of memory retention uses the chi-square test in non-parametric tests.

[0017] Further, in the S1 step, when in the indoor laboratory, the universal exhaust hood in the room needs to be opened at the same time to avoid the dispersion of the odor in the room, which leads to the failure of the experiment; when in the outdoor experiment, only the exhaust fan on the box needs to be opened.

[0018] The film has good air impermeability and hydrophobicity, etc. The insect antennae are installed and fixed upward. The size of the isolation chamber can be determined according to the size of the insect antennae and the convenience of operation. It is composed of a light transparent plate and a wrapped film, one end of the rectangular structure is a circular hole, which is just sleeved on the wall of the circular pipeline of the odor releaser and is fixed, and the other end opposite to it is not equipped with a transparent plate as an air outlet, keeping the air passage unobstructed, and the bottom surface of the rectangular structure is also not equipped with a light transparent plate, but is wrapped with a special film to form a bottom surface,

[0019] The micropore supports the upward lifting and fixing of the antennae, and the isolation chamber isolates the space where the tiny antennae (olfaction) of the insect head are located from the space where other head organs (such as tiny proboscis) are located, which can not only ensure that the antennae can only receive the stimulation of the conditioned odor, but also block the diffusion of chemical odor as an unconditioned appetite stimulating substance to the antennae. The gap between the micropore and the antennae is sealed with high-vacuum silicone grease, and special attention should be paid to not causing damage to the insect antennae during operation, otherwise it will affect the subsequent experiment; because the antennae and the proboscis are both on the head, the distance between them is very close, so the space for manual operation is very small.

[0020] Compared with the prior art, the present application has the following technical effects:

[0021] The present application has a system for simultaneously testing the olfactory conditioned proboscis extension reflex of insects driven by volatile or non-volatile appetite substances, and further uses the system to establish a training paradigm of conditioned PER driven by volatile unconditioned appetite stimulation. This provides a new tool for global scientists and technical personnel to evaluate the combined learning and memory condition of CS (conditioned odor) -US (volatile unconditioned stimulus) of insects, thereby leading and opening up a new research direction in the world. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1The structural schematic diagram of the device for detecting insect learning and memory; wherein, 1 - box, 11 - air pump, 12 - air filter, 13 - integrated air humidifier / flow meter, 14 - odor source chamber, 15 - odor release pipe, 16 - isolation chamber, 17 - insect body fixer, 18 - micropore, 19 - exhaust fan, 20 - adjustable fixing frame with clamp, 171 - fixed small tube, 172 - hole support, 21 - white light, 22 - infrared light.

[0023] Figure 2 The flow chart of the method for detecting insect conditioned proboscis extension reflex (learning and memory) based on the device of the present application.

[0024] Figure 3 The comparison chart of olfactory learning and memory of male Bactrocera dorsalis driven by volatile methyl eugenol (ME) and non-volatile sucrose respectively. (A) Comparison chart of olfactory learning performance of male Bactrocera dorsalis driven by ME and sucrose respectively. (B) Comparison chart of memory retention performance of ME and sucrose. DETAILED DESCRIPTION

[0025] Example 1: A device for detecting insect learning and memory

[0026] An experimental device for detecting insect learning and memory, the device comprising a box 1 made of opaque material, a pull-out door provided on the front of the box, an odor delivery device and a conditioning training device provided in the box; one end of the odor delivery device is connected to an air pump 11 outside the box through a hose, the other end is connected to an air filter 12, an integrated air humidifier / flow meter 13, an odor source chamber 14 and an odor release pipe 15 in sequence through a hose, the conditioning training device is composed of an isolation chamber 16 and an insect body fixer 17, the isolation chamber 16 has a hexahedral structure with an open right face as an air outlet, the odor release pipe 15 penetrates through the left face of the isolation chamber 16 and forms an angle of 15-45 degrees with the bottom face of the isolation chamber 16 (the isolation chamber must be kept horizontal to facilitate the operation of the insect body and antennae), the left, front, rear and upper faces of the isolation chamber 16 are composed of light transparent thin plates, the bottom face is wrapped with a film, a micropore 18 is provided in the center of the bottom film, the insect body fixer 17 is located below the micropore of the isolation chamber, an exhaust fan 19 is provided on the right wall of the box. The insect body fixer 17 is a fixed small tube 171 with a backrest and a hole support 172 for stabilizing the fixed small tube. The odor release pipe 15 is a hard pipe with a diameter ≤10 mm. A white light 21 and an infrared light 22 are further provided on the top of the box, the isolation chamber has a size of 40*30*30 mm, and the box has a size of 60*40*40 cm. An adjustable fixing frame 20 with a clamp for fixing the odor release pipe is further provided on the top of the box. A universal exhaust hood is further provided outside the box to avoid the accumulation of odors in the room.

[0027] Method for detecting insect olfactory condition PER using the above device

[0028] I. Experimental insects

[0029] The experimental B. dorsalis was collected in Yuanjiang County, Yunnan Province in July 2021. The mature fruit flies collected in the wild were placed in a rearing cage and reared in a 35x30x30cm rearing cage. The rearing environment had an indoor temperature of 25±2°C, a relative humidity of 50-70%, and a light cycle of 12h:12h (8:00-20:00 as the light period, and 20:00-8:00 as the dark period). A culture dish (15cm in diameter) filled with cotton soaked in 15% sucrose solution and yeast was placed in the cage for the fruit flies to eat. When the fruit flies matured, mango green fruits were placed in the cage for female B. dorsalis to lay eggs and for larvae to feed and develop. After 2-3 days, the mangoes containing eggs were placed in a sand tray lined with fine sand, and the mature larvae pupated in the moist fine sand. After 2-3 days, the eggs were sieved out of the sand tray and placed in a small culture dish, which was then placed in a large culture dish filled with moist cotton in a rearing cage. The eggs were allowed to hatch and the time was noted. After hatching, the fruit flies were placed in a sucrose solution and yeast (3:1) and allowed to mature (10-20 days after hatching) for behavioral experiments.

[0030] II. Experimental instruments and reagents

[0031] Experimental reagents: sucrose (Sucrose, Sigma-Aldrich), methyl eugenol (ME, ≥98%, Sigma-Aldrich), paraffin oil (Sigma-Aldrich), citral (95%, Sigma-Aldrich), activated carbon (Henan Coconut Carbon Environmental Materials Co., Ltd.), PBS solution (sodium chloride, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dihydrate, AR, all from Sigma-Aldrich), dimethyl sulfoxide (DMSO, ≥99.9%, Sigma-Aldrich).

[0032] III. Experimental methods

[0033] This experiment used volatile ME solutions of 0.029M (0.5 μg / μL) and 0.116M (2 μg / μL) as unconditioned appetite stimuli (US). Simultaneously, 0.029M (2 μg / μL) and 0.438M (150 μg / μL) sucrose solutions were used as another group of unconditioned appetite stimuli. Citral was selected as the conditioned stimulus (CS). Sucrose is a recognized natural reward substance; in conventional classic olfactory conditioned proboscis reflex experiments, sucrose solution is usually considered representative of food and participates in the experiment as an unconditioned stimulus. It should be noted that many plants in nature produce large quantities of volatile secondary metabolites. This experiment tested whether methyleugenol, a volatile plant secondary metabolite, could serve as an unconditioned stimulus to drive the establishment (learning and memory) of olfactory conditioned PER in insects.

[0034] S1 Adaptation Phase: The day before the experiment, select male oriental fruit flies with good activity and similar body size from the rearing box, place them in 50ml perforated centrifuge tubes containing water, and starve them for 12 hours; on the day of the experiment, place the insects in... Figure 1 In the insect body fixation device shown, the tiny antennae of the insect are carefully inserted into the micropores at the bottom of the isolation chamber, keeping the antennae vertically upward and extended. The gaps are then sealed with high-vacuum silicone grease (which both fixes the antennae and ensures airtightness). Special care must be taken during the operation to avoid damaging the insect's antennae, otherwise it will affect subsequent experiments (because the antennae are very small and easily broken, and the distance between the antennae and the proboscis on the insect's head is very close, so care must also be taken not to damage the proboscis). During operation, special care should be taken to avoid excessive pressure on the insect's head by the membrane (the membrane thickness needs to be controlled at ≤30μm), otherwise it will affect subsequent experiments. After 1 hour of acclimatization, gently touch the forelegs of the fruit fly in the device with absorbent cotton soaked in pure water and observe whether it exhibits a proboscis reflex. Remove any insects that still extend their proboscis after contact with pure water, and use the remaining insects for subsequent experiments. All conditioning training processes are conducted under infrared light. Take 15μL of citral (1:100) and place it on filter paper (4×1cm), then place it in the odor source chamber. Turn on the odor delivery device to provide odor stimulation. The odor in the filter paper of the odor source chamber is blown towards the fruit fly's antennae by the airflow (1L / min). At the same time, open the side wall of the box and set up multiple exhaust fans to immediately blow away the various odors, keeping them away from the insect's body to ensure the success of the experiment. Replace the filter paper after each training session.

[0035] The stages of S2 learning and training:

[0036] 1. Olfactory conditioning PER training method driven by the volatile secondary metabolite methyleugenol (ME)

[0037] Adopting such Figure 1The device was used to provide odor stimuli, air pump was turned on, charcoal filter was used, 15 μL of citral (1 : 100) was absorbed on filter paper (4 x 1 cm) and placed in the odor source chamber; the odor from the filter paper was blown by air flow (1 L / min) towards the proboscis of the fly. At the same time, the exhaust fan was turned on to immediately remove the odor from the body of the fly, ensuring the success of the test. After each training, the filter paper was replaced.

[0038] Development of the ME-driven olfactory conditioning PER training method (flowchart as Figure 2 ): After numerous trials, the design was to train each fly 5 times, with an interval of 10 min. Before the first training, the fly was adapted to the red light and air flow without odor for 15 s to reduce the disturbance caused by the change of new environment. After the adaptation, the odor stimulus (citral odor) was blown to the proboscis of male B. cucurbitae at a flow rate of 1 L / min. The flies that showed proboscis extension reflex to citral odor were excluded from the first training. After the odor was presented for 3 s, the fly was given 0.116 M or 0.029 ME solution as a reward. The fly was lightly touched on the proboscis with a piece of absorbent cotton soaked in the ME solution, which was removed after 3 s of continuous supply. The exhaust hood was turned on to remove the odor around the fly. The learning was evaluated by the proboscis extension reflex to the citral air flow. Only the flies that showed proboscis extension reflex within 3 s of air flow were considered to have learned the association between the conditioned stimulus (CS: citral odor) and the unconditioned ME stimulus. These flies were recorded as "learned", and the other flies were recorded as "unlearned". After the training, the flies were taken out of the device and placed in a room under an infrared lamp, with a temperature of 25-26 °C, a relative humidity of 50-60%, and a small amount of sucrose solution was fed every 1 h to prevent the flies from dying of excessive hunger.

[0039] 2. Sucrose-driven olfactory conditioning PER training method based on the device of the present application (flowchart as Figure 2) : The flies were put into the device for testing, and the specific operation was still the same as above, which proved that the device was also feasible in the case of non-volatile material sucrose. Each fly was trained for 5 times, and each training interval was kept at 10 min. Before the first training, the flies were adapted to the red light and odorless airflow for 15 s to reduce the interference brought by the change of new environment. After the adaptation, the olfactory stimulus (citral odor) was blown to the antennae of male B. cucurbitae at a flow rate of 1 L / min, and the flies that showed proboscis extension reflex to the citral odor were excluded in the first training. After the odor was presented for 3 s, the flies were given 0.116 or 0.438 M sucrose solution as a reward, and the proboscis of the flies was lightly touched with the sucrose solution soaked in the defatted cotton. After the solution and odor were removed, the exhaust hood was opened, and the surrounding odor was removed. The learning was evaluated by the proboscis extension reflex to the citral airflow, and only the flies that showed the proboscis extension reflex within 3 s of airflow were considered to have learned the association between the conditioned stimulus (CS: citral odor) and the unconditioned sucrose stimulus. These flies were recorded as "learned", and the other flies were recorded as "unlearned". After the training, the flies were taken out of the device and placed in a room under an infrared lamp, with a temperature of 25-26°C and a relative humidity of 50-60%, and a small amount of sucrose solution was fed every 1 h to prevent the flies from dying of excessive hunger.

[0040] In the paired group learning (acquisition) training stage, the flies were trained for 5 times of conditioning, and the proboscis extension reflex of the flies to the citral odor was recorded every time.

[0041] In the unpaired three control groups learning training

citral-only group, ME-only group, sucrose-only group

[0042] S3 After the end of the S3 memory retention learning training, the flies were removed from the device and placed in a room under an infrared lamp, maintaining a temperature of 25-26°C and a relative humidity of 50-60%, and fed with a small amount of sucrose solution every 1 h to avoid excessive starvation of the flies; then, the memory retrieval experiment was performed at 10 min, 24 h and 48 h, respectively. Flies that showed proboscis extension within 3 s of odor presentation were considered to have learned the association between the conditioned stimulus (CS: citral odor) and the unconditioned methyl eugenol stimulus and were recorded as “learned”; other flies were recorded as “not learned”;

[0043] S4 Statistical analysis The data of the olfactory conditioned proboscis extension reflex experiment were statistically analyzed using dichotomous method (correct PER: 1, incorrect or no PER: 0). The raw data were entered into Excel, and data analysis was performed using IBM SPSS Statistics 26, and graphs were plotted using OriginPro 2021; the performance changes caused by the number of training within a single learning curve were detected using Cochran Q; the comparison between groups of learning curves was analyzed using repeated measures ANOVA; the intergroup differences in memory retention were analyzed using the chi-square test in non-parametric tests. When comparing the data of each group, p < 0.05 indicates a significant difference, and p < 0.01 indicates a very significant difference, which is indicated by “a”, “b”, “c” or “*”, “**”.

[0044] 1.3 Experimental results

[0045] 1.3.1 Evaluation of the ability of volatile ME-driven insect olfactory learning and memory based on the device

[0046] To evaluate whether the volatile chemical methyl eugenol (ME) can drive associative learning in male B. dorsalis, the experimental results were analyzed, as shown in Figure 3 A, after 5 learning times, 66.7% of the flies in the 0.116M (0.5 pg) ME group and 63.3% of the flies in the 0.029M (2 pg) ME group formed olfactory conditioned PER. The results showed that male B. dorsalis successfully learned associative learning driven by two concentrations of ME; there was also no significant difference between the 0.029M (2 pg) ME group and the 0.116M (0.5 pg) ME group after 5 learning times.

[0047] The experimental results of the effect of ME on memory are shown in Figure 3 B, after the end of the training, the 10 min, 24 h and 48 h memory performances of the 0.116M (0.5 pg) ME group were 56.67%, 46.67% and 20%, respectively, and those of the 0.029M (2 pg) ME group were 56.67%, 43.33% and 16.67%, respectively.

[0048] These results show that the device can assess volatile ME-driven olfactory learning and memory in insects.

[0049] 1.3.2 Non-volatile sugar water-driven olfactory learning and memory ability based on the device

[0050] As Figure 3 A, 0.116 M (0.5 μg) sucrose group and control group of flies did not learn the olfactory association. But when the concentration was increased to 0.438 M (150 μg), 60% of the flies in the sucrose group learned the olfactory association.

[0051] As Figure 3 B, 10 min, 24 h, 48 h memory scores of 0.438 M (150 μg) sucrose group were 46.67%, 16.67%, 10%, respectively. These results show that the device can assess sucrose-driven olfactory learning and memory in insects.

[0052] 1.3.3 Comparison of non-volatile sucrose and volatile ME-driven olfactory learning and memory ability based on the device.

[0053] Further, the inventors compared the learning scores driven by sucrose and ME, and compared the scores of the last training. As Figure 3 A shows that there was no significant difference between 0.029 M (2 μg) ME group and 0.116 M (0.5 μg) ME group after 5 times of learning. However, 0.029 M sucrose group of flies could not form olfactory conditioned PER, and only 0.438 M (150 μg) sucrose group of flies formed olfactory conditioned PER, and there was no significant difference between 0.029 M ME group and 0.116 M ME group. This shows that 0.029 M and 0.116 M micro-amount of ME can reach the effect of 0.438 M sucrose. These results show that under the same micro-amount conditions, volatile ME-driven olfactory learning in insects is stronger than sucrose.

[0054] Next, the effects of the two solutions on memory were evaluated, and those that did not learn the olfactory association were not subjected to this experiment. First, the effect of ME solution on memory was evaluated, as Figure 3B shows that the 10 min, 24 h, 48 h memory scores of 0.116 M (0.5 μg) ME group were 56.67%, 46.67%, 20% respectively, and the 10 min, 24 h, 48 h memory scores of 0.029 M (2 μg) ME group were 56.67%, 43.33%, 16.67% respectively. The effect of sucrose solution on memory was also evaluated, and the 10 min, 24 h, 48 h memory scores of 0.438 M (150 μg) sucrose group were 46.67%, 16.67%, 10% respectively. No death was observed during the experiment. Chi-square test showed that there was no significant difference between 0.438 M sucrose group and 0.029 M ME group or 0.116 M ME group in 10 min short-term memory, and there was no significant difference between the two different ME concentrations. The 24 h long-term memory of 0.438 M sucrose group was significantly lower than that of 0.029 M ME group (p = 0.017) and 0.116 M ME (p = 0.010) group, and there was no significant difference between the two different ME concentrations (p = 0.697). The 48 h long-term memory of 0.438 M sucrose group was not significantly different from that of 0.029 M ME group (p = 0.390) and 0.116 M ME (p = 0.214) group, and there was no significant difference between the two different ME concentrations (p = 0.660). These results showed that even 0.438 M sucrose could not enhance the 24 h long-term memory of B. cucurbitae. However, 0.116 M and 0.029 M micro-amount of ME could enhance the 24 h long-term memory of B. cucurbitae. Therefore, volatile ME-driven insect olfactory memory is more effective than sucrose.

Claims

1. An experimental apparatus for detecting insect learning and memory, characterized in that: The device includes a box (1) made of opaque material, with a pull-out door on the front. An odor delivery device and a conditioning training device are installed inside the box. The odor delivery device is an air pump (11) located outside the box and connected via a hose. The air pump (11) is connected in sequence via a hose to an air filter (12), an integrated air humidifier / flow meter (13), an odor source chamber (14), and an odor release pipe (15). The conditioning training device consists of an isolation chamber (16) and an insect body fixation device (17). The isolation chamber (16) has a hexahedral structure with an opening on the right side, the opening being the air outlet. The odor release pipe... (15) Penetrates the left side of the isolation chamber (16) and forms an angle of 15-45 degrees with the bottom of the isolation chamber (16). The left, front, back and top of the isolation chamber (16) are composed of lightweight transparent thin plates, and the bottom is covered with a film. A micro hole (18) is set in the center of the bottom film. The insect body fixation device (17) is located below the micro hole (18) of the isolation chamber. An exhaust fan (19) is set on the right side wall of the box. The insect body fixation device (17) is a fixed tube (171) with a backrest and a perforated bracket (172) for stabilizing the fixed tube. A white light (21) and an infrared light (22) are also set in the top of the box (1).

2. The experimental apparatus for detecting insect learning and memory according to claim 1, characterized in that: The odor release tube (15) is a rigid tube with a diameter ≤10 mm.

3. The experimental apparatus for detecting insect learning and memory according to claim 1, characterized in that: The box (1) is 60 * 40 * 40 cm in size, and the isolation chamber is 40 * 30 * 30 mm in size.

4. The experimental apparatus for detecting insect learning and memory according to claim 1, characterized in that: The top of the box is also equipped with an adjustable fixing bracket (20) with clamps for fixing the odor release tube (15).

5. The experimental apparatus for detecting insect learning and memory according to claim 1, characterized in that: The enclosure is also equipped with a universal exhaust hood.

6. A method for detecting insect learning and memory using the device according to any one of claims 1-5, comprising the following steps: The day before the S1 adaptation phase experiment, male oriental fruit flies with good activity and similar body size were selected from the rearing box and placed in a 50ml perforated centrifuge tube containing water for 12 hours of starvation. On the day of the experiment, the insects were placed in an insect body fixation device, and the tiny antennae of the insects were carefully guided into the micropores of the membrane at the bottom of the isolation chamber, keeping the antennae vertically upward and exposed, and the gaps were carefully sealed with high-vacuum silicone grease. After 1 hour of adaptation, the forelegs of the fruit flies in the device were gently touched with absorbent cotton soaked in pure water to observe whether they exhibited a proboscis reflex. Insects that still extended their proboscis after contact with pure water were removed, and the remaining individuals were used for subsequent experiments. All conditioning training processes were carried out under infrared light. The conditioned stimulus odorant was aspirated, placed on filter paper, and placed in the odorant chamber. Turn on the air pump, and the odor in the filter paper of the odor source chamber is blown towards the antennae of the fruit fly by an airflow of 1 L / min. At the same time, open the side wall of the box and set up multiple exhaust fans to immediately blow away the various odors and keep them away from the insect's body to ensure the success of the experiment. Replace the filter paper after each training session. Before the first training session, the S2 conditioning training involved 15 seconds of airflow adaptation to reduce interference from the new environment and minimize insect stress. After adaptation, the odor was blown towards the antennae of male oriental fruit flies at a flow rate of 1 L / min. Flies exhibiting a proboscis reflex to the odor were excluded during the first training session. Three seconds after the release of the conditioned stimulus odorant, the flies were rewarded with an appetite stimulant solution. A soft, absorbent cotton ball soaked in the appetite stimulant solution was gently touched to the fly's proboscis for 3 seconds. The odor release was maintained for 6 seconds. In the CS-US paired group training, the flies underwent a total of 5 conditioning training sessions, with a 10-minute interval between each session. The proboscis reflex to the odorant was recorded in each experiment. The appetite stimulant solution included volatile drugs and non-volatile foods. After the S3 memory retrieval training, the fruit flies were carefully removed from the isolation room and placed in a room under infrared lamps, maintaining a temperature of 25-26℃ and a relative humidity of 50-60%. They were fed small amounts of sucrose solution every hour to prevent starvation and death. Then, memory retrieval experiments were conducted at 10 minutes, 24 hours, and 48 hours. Fruit flies that exhibited a proboscis reflex within 3 seconds of odor presentation were considered to have remembered the relationship between the conditioned stimulus and the unconditioned appetite stimulus and were recorded as "learned." Other fruit flies were recorded as "not learned." S4 statistical analysis used the dichotomy method to statistically analyze the experimental data of olfactory conditioned beak extension reflex; the raw data were entered into Excel, and data analysis was performed using IBM SPSS Statistics 26, and graphs were generated using Origin Pro 2021; The performance variation caused by the number of training sessions within a single learning curve was measured using the Cochran Q test; the inter-group comparison of learning curves was performed using repeated measures ANOVA; and the inter-group differences in memory retention were measured using the chi-square test, a non-parametric test.

7. A method for detecting insect learning and memory using the device according to claim 6, characterized in that: In step S1, when conducting experiments in an indoor laboratory, the universal exhaust hood in the room needs to be turned on simultaneously to prevent odors from spreading indoors and causing the experiment to fail; when conducting experiments outdoors, only the exhaust fan on the chamber needs to be turned on.

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