Use of methyleugenol in the preparation of a product for rapidly improving the locomotor ability of fruit fly insects

Abstract

The present invention belongs to the field of agricultural insect behavior, and particularly relates to the use of methyl eugenol in the preparation of a product for rapidly improving the locomotor ability of fruit fly insects. Experiments of the present invention have proved that low-dose methyl eugenol can rapidly improve the locomotor level of fruit fly insects, including crawling, flying, etc. Therefore, the present invention protects the application that the exposure to low-dose methyl eugenol can immediately and rapidly improve the locomotor ability of male Bactrocera dorsalis, which solves the problems that in the application of the SIT technology system for Bactrocera dorsalis, the insect body is damaged by nuclear radiation, the locomotor ability decreases, and the diffusion level is low, thus affecting the efficiency and cost of the SIT technology. The present invention provides a key scientific basis for the innovative upgrade application of methyl eugenol in the SIT prevention and control technology, and also opens up a new way for the innovative upgrade of the global pest SIT technology system.

Description

Technical Field

[0001] The present invention relates to the use of methyleugenol in the preparation of a product for rapidly improving the motor ability of fruit fly insects, and belongs to the technical field of agricultural insect behavior. Background Art

[0002] The oriental fruit fly (Bactrocera dorsalis Hendel) belongs to the genus Bactrocera of the family Tephritidae in the order Diptera. Female adult oriental fruit flies lay eggs under the fruit epidermis, with 10 - 30 eggs laid each time. The larvae feed on the pulp, causing the fruit to rot or drop, thus seriously affecting the fruit yield and quality. This insect can damage the fruits of a variety of fruit trees (such as citrus, mango, peach, pomegranate, jujube, pear, apple, etc.) and vegetables (such as eggplant, pepper, cucumber, tomato, etc.). After being damaged by this insect, the fruit shrivels, shrinks and rots, forming "maggot fruits", causing a large number of fruit drops, seriously affecting the yield, and even leading to a complete crop failure.

[0003] The invasion and spread of the oriental fruit fly have continuously had a huge impact on the development of the fruit and vegetable industries in China and the world. In order to control its damage to the greatest extent, comprehensive control measures have been developed, such as chemical, biological, physical, agricultural, sterile insect technique (SIT) and other comprehensive control measures. SIT is a green and environmentally friendly technology, which refers to the large-scale release of sterilized male pests caused by nuclear radiation in the field. They mate with wild female insects but have no offspring, thus reducing the population of pests in a certain area. It has been used to control pests including mosquitoes, fruit flies (Mediterranean fruit fly, Mexican fruit fly, oriental fruit fly, melon fly, etc.). The benefits of using this technology include: significantly reducing crop and livestock production losses; providing conditions for exporting commodities to high-value markets without quarantine restrictions; and protecting the environment by reducing the use of pesticides. However, the SIT technology is a complex technology that requires the establishment of large factories, involving large-scale breeding and production facilities and technologies for male and female insects, large-scale separation technology for male insects, male radiation sterilization technology, large-scale release technology in the target area, and so on. After these sterilized male insects mate with wild female insects, they will not produce offspring, thus reducing the population of this pest. Each link factor in production, including nuclear radiation treatment, as well as transportation and release links, may affect the effectiveness of the SIT technology. A series of production operations such as nuclear radiation, in addition to causing sterilization, will also inevitably lead to a certain degree of decline in the motor activity of male insects to some extent. This will cause male insects not to spread rapidly in the release area, which increases the application cost of this technology and restricts the maximization of the efficiency of the SIT technology. Therefore, in order to improve the efficiency of SIT, it is necessary to improve the production quality of male insects, including improving the motor ability of insects, etc.

[0004] Numerous studies have fully confirmed that movement is the basis for animals to adapt to complex and changing environments, search for food, avoid natural enemies, compete for and expand habitats, and reproduce offspring. In large-scale breeding facilities, ensuring that the locomotor ability of male fruit flies does not decline significantly is a key technical task in production quality control. If the locomotor ability of male fruit flies can be significantly improved, it is a huge technological advancement. Similarly, in the release stage in the target area, ensuring that the locomotor ability of the mass-released male fruit flies does not decline significantly is also a key technical task in production quality control. If the locomotor ability of male fruit flies can be significantly improved during release, it is a huge technological advancement. This is because male fruit flies with low locomotor activity may not even be able to ensure their own foraging and survival, let alone compete with wild male fruit flies and engage in courtship and mating activities with females. Therefore, in the SIT technology system, in the breeding stage and the release stage, if the locomotor ability of male insects can be rapidly improved, it can naturally enhance the survival adaptability of male insects and also reduce the cost rate, which is of great significance for the effectiveness of the SIT program. However, so far, there is no solution or approach for "effectively enhancing locomotor activity" in the entire global SIT technology system.

[0005] How to improve the locomotor ability of fruit flies such as Bactrocera dorsalis? We attempted to break through this technical problem. A large number of previous indoor and field studies have shown that male Bactrocera dorsalis can be strongly attracted by the odor of methyl eugenol and are also willing to ingest it. Methyl eugenol (ME) is a phenylpropanoid chemical, also known as pentenyl methyl ether, 3,4-dimethoxy-4-(2-propenyl) benzene, etc. Methyl eugenol is an oily liquid, colorless to light yellow, with the odor of cloves and carnations. It is unstable at room temperature and volatile. In 1975, American scientists compared the attractiveness of methyl eugenol and 34 of its similar-structured compounds to male fruit flies and found that methyl eugenol is the most potent odor attractant compound. Since then, people have started to pay a great deal of attention to the application of methyl eugenol in fruit fly control. Methyl eugenol (ME), as a commonly used and highly effective male attractant, is widely used in the control of various fruit flies such as Bactrocera correcta, Bactrocera cucurbitae, and Bactrocera dorsalis. Methyl eugenol is usually used in the field for fruit fly population monitoring, odor attraction, etc., and can be an important supplement to chemical control methods for pest management. In other words, in the existing technology, methyl eugenol is usually used as a specific odor attractant for male fruit flies.

[0006] So far, there has been no report that methyl eugenol (ME) can rapidly improve the locomotor activity of male Bactrocera dorsalis, nor has the potential application of ME in the sterile insect technique (SIT) of B. dorsalis been explored. B. dorsalis has a very wide distribution range and causes extremely serious harm. If there is a method that can improve the locomotor activity of males, it will effectively improve the cost-effectiveness of the global SIT application technology. The inventors accidentally discovered that ME can improve the locomotor activity of B. dorsalis, and this discovery will be of great significance for improving the application efficiency of SIT technology. Summary of the Invention

[0007] In view of the above technical problems, the present invention provides the use of methyl eugenol in the preparation of a product for rapidly improving the locomotor ability of fruit fly insects.

[0008] Furthermore, the methyl eugenol is the only active ingredient.

[0009] Preferably, the single dose of the methyl eugenol is 1 - 3 μg / fly.

[0010] Preferably, the administration method of the methyl eugenol is the autonomous sucking of the insect mouthparts.

[0011] Preferably, the fruit fly insects include Bactrocera correcta and B. dorsalis that are sensitive to methyl eugenol.

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] The present invention confirms that 1 - 3 μg / fly of methyl eugenol can rapidly improve the upward climbing, horizontal crawling and flying abilities of B. dorsalis; the present invention fully proves that low-dose methyl eugenol can rapidly improve the locomotor activity of B. dorsalis, which not only breaks through the original understanding of the use of methyl eugenol by people and has very important scientific value, but also breaks through the SIT technology bottleneck. This breakthrough in technology bottleneck will provide a solid scientific basis for the innovation and upgrading of the B. dorsalis SIT technology and have a profound impact on the improvement of the global SIT technology system. Brief Description of the Drawings

[0014] Figure 1 Shows the relationship between the mortality rate of B. dorsalis and the intake dose of methyl eugenol;

[0015] Figure 2 Shows the comparison chart of the crawling movement time of B. dorsalis in different ME dose groups before and after ME exposure; Figure 2 A shows the comparison chart of the crawling movement time of B. dorsalis before and after treatment with 0 - 1035 μg; Figure 2 B is Figure 2 An enlarged view of 0 - 4 μg in A;

[0016] Figure 3Figure for the increase in the flight activity of male *Bactrocera dorsalis* by 2 μg ME, where Figure 3 A is a comparison graph of the rapid increase in the average flight speed of male *Bactrocera dorsalis* within 15 min after ME exposure; Figure 3 B is a comparison graph of the cumulative flight time of male *Bactrocera dorsalis* within 15 min after ME exposure; Figure 3 C is a comparison of the increase in the cumulative flight distance of male *Bactrocera dorsalis* within 15 min after ME exposure. Specific implementation method

[0017] Implementation materials and instruments

[0018] 1. Experimental insects

[0019] Insect rearing: The *Bactrocera dorsalis* used in the experiment was collected from Yuanjiang, Yunnan in July 2021. The *Bactrocera dorsalis* (10 - 15 d) was reared indoors at a temperature of 25 ± 2 °C, a relative humidity of 50 - 70%, and a photoperiod of 12 h:12 h (Wee et al., 2007; Martha et al., 2018) (the light period was from 8:00 to 20:00, and the dark period was from 20:00 to 8:00). Fresh green mangoes were provided for *Bactrocera dorsalis* to lay eggs and for larvae to feed and develop. The mangoes with eggs were placed in a sand tray covered with fine sand, and the mature larvae pupated in the moist fine sand. The pupae were placed in a petri dish (diameter 15 cm) containing moist cotton and placed in a rearing box until they emerged. Gender identification was carried out within 3 days after emergence, and males and females were reared separately in different cages (35 × 30 × 30 cm) to prevent mating. Two petri dishes were placed in the box, containing yeast powder and sugar water respectively for the fruit flies to eat. Yeast powder and sugar water were provided respectively for the fruit flies to eat.

[0020] 2. Experimental instruments and reagents

[0021] Experimental reagents: Methyl eugenol (ME, ≥98%, Sigma), DMSO (Sigma), yeast (Sigma - Aldrich), sucrose (Sigma - Aldrich), pure water.

[0022] Instrument equipment: Ice maker, stereomicroscope (OLYMPUS - SZX16), micromanipulator (WPI - M3301), capillary (Drummond Wiretrol 1 - 5 uL), 1 ml syringe, glass finger - shaped tube (h = 10 cm, d = 2 cm), petri dish (d = 6 cm), flight mill (FXM - Z, Jiaduo Industry and Trade Co., Ltd.), copper wire, 502 glue, insect pins, etc.

[0023] 3. Data statistics and analysis

[0024] All data are expressed as mean ± S.E.M., and P < 0.05 is considered statistically significant. The original data were entered into Excel and analyzed using IBM SPSS Statistics 26, and graphs were plotted using Origin Pro 2021 and Graphpad prism. For the vertical climbing time, flight distance, cumulative flight time, and average flight speed that conform to a normal distribution, analysis of variance was used for testing, otherwise, non-parametric tests with multiple comparisons (Kruskal-Wallis test) were used.

[0025] Example 1: Effects of Different Doses of Methyl Eugenol on the Mortality of Male Bactrocera dorsalis

[0026] Establishment of a Precise Quantitative Drug Administration Method for Fruit Fly Individuals. Gently fix unmated, sexually mature male fruit flies with a 200 μL pipette tip, exposing the heads of the fruit flies to ensure their free movement and avoid damaging their bodies; use capillary action to suck pure ME liquid into a glass capillary, and precisely control the glass capillary with a micro-manipulator with an accuracy of 1 μm. Under a microscope, use an electric micro-manipulator to place one end of the capillary about 3 - 5 mm from the mouthparts of the fruit fly. When the fruit fly senses the ME, it will actively extend its proboscis and lick the ME. This method reduces the damage to the fruit fly caused by direct contact with the feeding tube. As the fruit fly extends its proboscis to lick the liquid in the feeding tube, the liquid level in the glass tube gradually drops, and the movement and position of the liquid level are observed and measured. After the fruit fly has completed feeding on ME at the corresponding scale (dose), immediately remove the glass capillary.

[0027] To observe the effects of acute ingestion of different doses of ME on the survival rate of male Bactrocera dorsalis, the survival rate of Bactrocera dorsalis 4 h after ingestion of ME (a commonly used evaluation method for toxicity measurement) was determined. Male Bactrocera dorsalis (10 - 15 days old) with similar body sizes and active movements were selected for the experiment. Through the above drug administration method, after the male Bactrocera dorsalis ingested different doses of ME, they were placed in a petri dish and observed for 4 h, and the survival and death conditions at different doses within 4 h were recorded, and the mortality rate was calculated.

[0028] As Figure 1As shown, when male Bactrocera dorsalis were respectively fed 0 (negative control group), 2, 10, 20, 50, 300 μg of ME, the mortality rate within 4 h was 0; however, when male Bactrocera dorsalis were respectively fed 400, 500, 1035 μg of ME, the mortality rate within 4 h was 100%. The above results indicate that low-dose ME intake does not affect its survival, but high-dose intake is lethal. This is consistent with the research results of the existing technology. At the same time, the total content of the main decomposition products (2-allyl-4,5-dimethoxyphenol, trans-coniferyl alcohol) of ME metabolism in the rectal gland of Bactrocera dorsalis on wild orchids can reach 50 μg (Nishida et al., 2004; Tan et al., 2002; Tan et al., 2006), which shows that the amount of ME ingested by a single Bactrocera dorsalis in the wild can be higher than 50 μg.

[0029] It shows that the low-dose ME in the present invention can ensure the survival of Bactrocera dorsalis.

[0030] Example 2 Effect of low-dose ME on the locomotor ability of Bactrocera dorsalis

[0031] 2.1 Effect of low-dose ME on the vertical climbing movement of Bactrocera dorsalis

[0032] Male Bactrocera dorsalis aged 10 - 15 d, with similar individual sizes and relatively active movements, were selected from the breeding cage for the experiment. The experimental time was from 9:00 to 13:00 in the morning. The specific scheme was as follows: After taking out the male insects to be tested, they were placed in a centrifuge tube, anesthetized with ice, weighed, and then the experiment was carried out. After being anesthetized by freezing, the fruit flies were loaded into a 200 μL pipette tip. ME liquid with a specified dose was aspirated into a capillary tube (Drummond Wrietrol 1 - 5 μL). With the help of a micro-manipulator (M3301R, USA) with an accuracy of up to 1 μm, the glass capillary tube was precisely controlled. Under a SZX16 stereomicroscope (Olympus, TOKYO, Japan), one end of the capillary tube was placed about 3 - 5 mm away from the mouthparts of the fruit fly using the micro-manipulator. The fruit fly would actively extend its proboscis and lick the ME. After the feeding was completed, the fruit fly was carefully removed from the pipette tip to avoid any damage to the body. Then it was transferred into a finger-shaped glass tube device (2 cm in diameter and 10 cm in height) for testing the climbing ability of the fruit fly. First, it was allowed to adapt in the tube for 5 min. Due to negative geotaxis, the insects would climb to the upper part of the tube. The glass tube was gently shaken to make them return to the bottom of the glass tube again. The time required (climbing time) for each group of fruit flies to climb from the bottom to the middle position of the top lid on the vertical plane was recorded. Each sample was observed 5 times repeatedly, and the interval time between each test was 1 min. The total time for each single observation of each sample was set to 15 min.

[0033] The results are as Figure 2As shown in A, when Bactrocera dorsalis was acutely exposed to 1, 2, and 3 μg ME respectively, the vertical climbing times of these three dose groups were significantly lower than those of the control group (8.02 ± 0.11 vs. 12.35 ± 0.25 s; 7.42 ± 0.09 vs. 12.35 ± 0.25 s; 7.91 ± 0.18 vs. 12.35 ± 0.25 s, p < 0.001, n = 30 for each group). This indicates that the vertical movement ability of Bactrocera dorsalis was enhanced by ME. When male Bactrocera dorsalis was acutely exposed to a dose of 4 μg, there was no significant difference in the vertical climbing time compared with the control group (11.12 ± 0.57 vs. 12.35 ± 0.25 s, p > 0.05, n = 30), and the vertical climbing time was higher than that of the 1, 2, and 3 μg ME groups (11.12 ± 0.57 vs. 8.02 ± 0.11 s, 11.12 ± 0.57 vs. 7.42 ± 0.09 s, 11.12 ± 0.57 vs. 7.91 ± 0.18 s, p < 0.001). When Bactrocera dorsalis was exposed to doses of 10, 20, 50, 100, and 300 μg ME respectively, the vertical climbing times of these 5 groups were all significantly higher than those of the control group (18.78 ± 0.42 vs. 12.35 ± 0.25 s; 28.56 ± 0.95 vs. 12.35 ± 0.25 s; 47.01 ± 0.89 vs. 12.35 ± 0.25 s; 193.25 ± 1.65 vs. 12.35 ± 0.25 s; 483.37 ± 1.66 vs. 12.35 ± 0.25 s, p < 0.001, n = 30 for each group). When the intake of Bactrocera dorsalis reached 400, 500, and 1035 μg ME, it could not complete the climbing task within 15 min (n = 10 for each group), indicating that the motor activity of Bactrocera dorsalis was inhibited. The above results suggest that the effect of ME on the crawling ability of Bactrocera dorsalis is dose-dependent.

[0034] Meanwhile, the climbing time data of flies before ME exposure (self-control) and after ME treatment were further compared to more intuitively reflect the effect of ME. As Figure 2After treatment with 1, 2, and 3 μg ME, the vertical climbing time was lower than the self-baseline before ME exposure (8.02 ± 0.11 vs. 12.62 ± 1.37 s; 7.42 ± 0.09 vs. 12.05 ± 0.86 s; 7.91 ± 0.18 vs. 11.98 ± 0.57 s, p < 0.001, n = 30 for each group). After treatment with 4 μg ME, there was no significant difference in the vertical climbing time compared to the self-baseline before ME exposure (11.12 ± 0.57 vs. 12.62 ± 1.37 s; p > 0.05, n = 30 for each group). After treatment with 10, 20, 50, 100, and 300 μg ME, the vertical climbing time of these groups was significantly longer than the self-baseline before ME exposure (18.78 ± 0.42 vs. 11.89 ± 0.39 s; 28.56 ± 0.95 vs. 12 ± 0.95 s; 47.01 ± 0.89 vs. 13.33 ± 0.72 s, 193.25 ± 1.65 vs. 11.56 ± 0.98 s; 483.37 ± 1.66 vs. 14.86 ± 0.89 s, p < 0.001, n = 30 for each group). However, when the intake of Bactrocera dorsalis reached 400, 500, and 1035 μg ME, the flies in these groups did not climb (0 vs. 13.31 ± 0.98 s, 0 vs. 11.57 ± 0.95 s, 0 vs. 12.13 ± 0.78 s, p < 0.001, n = 30 for each group), and the flies lost their climbing ability. The above results further indicated that ME profoundly affected the crawling activity of Bactrocera dorsalis and showed a dose-dependence.

[0035] The bottom behavior of flies in different dose treatment groups before climbing the wall was further qualitatively observed. As shown in Table 1, after treatment with 0, 1, 2, 3, and 4 μg ME, before climbing the wall, the flies could still crawl back and forth at the bottom of the narrow tube (no difference could be distinguished). After treatment with 10, 20, 50, 100, and 300 μg ME, before climbing the wall, the crawling movement of the flies at the bottom of the tube became significantly slower. After treatment with 400 and 500 μg ME, the flies immediately stood still at the bottom of the tube and crawling disappeared. After treatment with 1035 μg ME, the flies immediately lost the ability to control their posture at the bottom of the tube, could not stand, and their bodies overturned.

[0036] In summary, low-dose ME increased the crawling activity of flies.

[0037] Table 1 Qualitative observation of the locomotor activity of male Bactrocera dorsalis at the bottom of the tube after acute ingestion of different doses of ME

[0038] dose (μg / fly) Behavior at tube bottom 0 ++ 1 ++ 2 ++ 3 ++ 4 ++ 10 + 20 + 50 + 100 + 300 + 400 ＝ 500 ＝ 1035 -

[0039] Note: ++ represents walking, + represents sluggish, = represents immobility, - represents loss of posture control

[0040] 2.2 Effect of low-dose ME on the flight movement of fruit flies

[0041] Adult male Bactrocera dorsalis with similar size and activity were placed in centrifuge tubes, numbered and weighed. Then, they were anesthetized with ice for 5 min. A suspension loop with a radius of 3 cm was made by oneself. The suspension loop was made by forming a copper ring with a diameter of 0.1 cm at one end of the copper wire, and the other end was wound 15 times around an insect pin 3.5 cm long. Dip a little 502 glue and stick it on the pronotum of the anesthetized Bactrocera dorsalis, confirm that the head, thorax, abdomen and base of the wings of Bactrocera dorsalis are not stained, stick the suspension loop on the pronotum, keep the suspension loop perpendicular to Bactrocera dorsalis, and gently blow air to make the glue solidify quickly. The insect pin needs to be connected with a small black paper (1.5×0.4 cm) first, which is used for induction and calculating the number of circles of the insect's flight, and then it is connected with the suspension loop.

[0042] After Bactrocera dorsalis woke up, gently blow air to observe its wing vibration, and eliminate individuals that could not vibrate their wings normally or had uneven wing vibration. After completing the above steps, different doses of ME were provided to Bactrocera dorsalis, and according to the numbers, they were placed between two micro magnets on the flight mill, ensuring that the fruit flies were placed horizontally for stable flight. Start the flight mill system and record the average flight speed, cumulative flight time and distance of the fruit flies. During the suspension flight, keep the indoor temperature at 25±1℃ and the relative humidity at 60%-70%, and the start time of the experiment is 9:00 am.

[0043] As Figure 3 shown: within 15 min after the 2 μg ME treatment group ingested ME, the average flight speed, cumulative flight time and cumulative flight distance were all significantly higher than those of the control group (p<0.001), indicating that the flight movement ability of Bactrocera dorsalis was enhanced by 2 μg ME. Figure 3 Figure for the improvement of the flight activity of male Bactrocera dorsalis by 2 μg ME, in which Figure 3 A is a comparison chart of the rapid increase in the average flight speed of male Bactrocera dorsalis within 15 min after ME exposure; Figure 3 B is a comparison chart of the cumulative flight time of male Bactrocera dorsalis within 15 min after ME exposure; Figure 3 C is a comparison of the increase in the cumulative flight distance of male Bactrocera dorsalis within 15 min after ME exposure.

[0044] In summary, the experiments of the present invention show that low-dose ME exposure immediately and rapidly enhances the locomotor activity of Bactrocera dorsalis. The research of the present invention shows that in the SIT technology system, especially in the reproduction stage and the release stage, 1-3 μg of ME can rapidly improve the locomotor ability of male insects, which can naturally improve the survival adaptability and reproductive mating ability of male insects, will be beneficial to reducing the cost of SIT and improving the efficiency of SIT, and has important significance for the effect of the SIT program.

Claims

1. Use of methyleugenol in the preparation of a product for rapidly improving the locomotor ability of fruit fly insects, characterized in that: The movement described is flight or crawling, and the dosage of methyl eugenol is 1 - 3 μg / fly.

2. The use according to claim 1, characterized in that, The methyl eugenol described is the only active ingredient.

3. The use according to claim 1, wherein The administration method of the methyl eugenol is ingestion by the insect mouthparts.

4. The application according to claim 1, characterized in that: The fruit fly insects described include the guava fruit fly and the oriental fruit fly that are sensitive to methyl eugenol.

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