Method for detecting feeding and oviposition selectivity of lepidoptera insects to transgenic Bt corn and conventional corn

Abstract

The invention provides a method for detecting feeding and oviposition selectivity of lepidoptera insects to transgenic Bt corn and conventional corn. According to the invention, a method for researching feeding selectivity and oviposition selectivity of lepidoptera pests such as beet armyworm to Bt corn and conventional corn and application thereof are adopted, so that feeding selection effect of larvae such as beet armyworm to Bt corn and conventional corn and difference of adult oviposition preference are determined; important data references are provided for environmental safety evaluation of unexpected effects and the like of transgenic Bt corn and formulation of pest Bt resistance treatment strategies. The method has remarkable advantages in the aspects of material selection, test design, feeding selectivity evaluation method and oviposition behavior observation, and is wide in application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of insect ecology, environmental safety assessment of genetically modified maize, and ecological risk control. Specifically, it relates to a method for detecting the selectivity of lepidopteran insects in feeding and oviposition on genetically modified Bt maize and conventional maize. Background Technology

[0002] Expressing Bacillus thuringiensis (Bt) Bacillus thuringiensis Genetically modified insect-resistant maize containing Bt toxin (Bt maize for short) is an important tool for integrated pest management targeting lepidopteran and coleopteran pests. Since its widespread adoption, Bt maize has significantly reduced pesticide use globally, increased crop yield and quality, and controlled major pests such as the European corn borer and fall armyworm in certain regions. Pilot industrial-scale plantings of Bt maize have demonstrated significant insect resistance, while also bringing considerable economic, social, and ecological benefits.

[0003] Since genetically modified maize typically carries foreign genes and expresses foreign proteins, the artificial insertion of these genes can alter the plant's original genome structure and endogenous metabolic pathways, thereby affecting the composition and content of secondary metabolites and the release profile of volatile organic compounds. These metabolic changes may have potential impacts on the growth, development, feeding behavior, oviposition selection, and population dynamics of herbivorous insects. Determining the effects of transgenic plants on the feeding and oviposition behaviors of herbivorous insects is crucial for revealing the metabolic changes in transgenic maize plants and scientifically assessing their ecological and environmental safety.

[0004] Meanwhile, with the long-term and widespread cultivation of Bt maize, the formulation and implementation of effective pest resistance management strategies are crucial for its sustainable development. Among these, the shelter strategy is one of the core measures to delay the development of pest resistance. In a planting system where Bt maize and conventional maize (shelter) coexist, the feeding and oviposition selection behaviors of pests on different plants directly affect their population dynamics and resistance evolution. On the one hand, the movement of larvae between adjacent plants may reduce their intake of Bt toxins, creating a functionally low-dose environment, thereby weakening the control effect of Bt maize and accelerating resistance evolution. On the other hand, the non-random oviposition behavior of female moths on both Bt maize and conventional maize (shelter) plants may increase the proportion of populations under selective pressure, further affecting the resistance management effectiveness of the shelter strategy. Therefore, in-depth research on the feeding and oviposition selection behaviors of target pests between Bt maize and shelter plants has significant scientific value for optimizing resistance management strategies.

[0005] Leaf disc method is a classic method for studying the feeding selection behavior of lepidopteran larvae, among which Jermy leaf disc method is the most commonly used. Tang Qingbo et al. optimized and improved this method, which improved the simplicity of operation and the intuitiveness of observation. However, when applied to the study of Bt corn feeding behavior, this method still has limitations: on the one hand, it mainly focuses on short-term feeding amount determination, lacks dynamic recording of changes in larval feeding behavior, especially it is difficult to assess the long-term feeding behavior of Bt corn on target pests; on the other hand, this method does not consider the potential impact of plant volatile compounds on larval behavior, and cannot reveal the changes in the avoidance response of pests before contacting plant tissues. Therefore, the existing leaf disc method cannot meet the needs of Bt corn metabolite effect evaluation.

[0006] In the study of oviposition selection behavior, cage method is commonly used. This method places different host crops in the same cage environment and releases a certain number of adults to freely oviposit to evaluate the selection preference. However, this method has the following limitations when applied to the study of Bt corn oviposition selection: first, it usually does not consider the difference in damage degree between Bt corn and conventional corn and the influence of larvae on adult oviposition selection, resulting in incomplete and systematic experimental results; second, this method usually only sets a 2-3 day oviposition observation period, ending the investigation before the larvae hatch, making it difficult to fully reflect the long-term oviposition selection dynamics of adult pests; in addition, the complex operation, poor repeatability, many interference factors, and difficult data collection of field cage experiments also limit the application of this method in large-scale experiments.

[0007] In recent years, some special devices have been used for the study of feeding and oviposition selection behavior of phytophagous insects, such as a device for observing insect oviposition selection, a device and method for detecting the feeding selection behavior of phytophagous insects, and "Y" type olfactometer. Although these devices have improved the controllability and data collection efficiency of the experiment, they still have limitations in studying the impact of Bt corn on the feeding and oviposition behavior of target pests and indicating the effect of Bt corn metabolites. For example, CN117178961A uses a trend tube, a trap plate, and a insect placement groove to study the feeding behavior of insects. Researchers need to repeatedly disassemble and assemble the equipment and place insects in designated locations, which is complex and can interfere with the natural behavior of insects, affecting the reliability of the results. CN219182500U guides insects to perceive volatile information from different plants for oviposition selection by setting two horizontally aligned boxes and a middle cylinder structure, but the fixed oviposition environment limits the free movement of insects between different host plants, which affects the ecological authenticity of the oviposition selection behavior in natural conditions. When studying the feeding behavior of insects using "Y" type olfactometer, only one type of insect can be tested for feeding preference on two test plants at a time, and only one insect can be placed in the device during the experiment, which is time-consuming and labor-intensive, and the data acquisition efficiency is low. SUMMARY

[0008] The present application aims to provide a method for detecting the feeding and oviposition selectivity of Lepidoptera insects to transgenic Bt corn and conventional corn.

[0009] To achieve the object of the present application, in the first aspect, the present application provides a method for detecting the feeding selectivity of Lepidoptera insects to transgenic Bt corn and conventional corn, comprising the following steps: (1) A wet filter paper with the same size as the bottom of a 10-20 cm diameter culture dish is laid on the bottom of the dish, and the filter paper is equally divided into eight units with the center of the filter paper as the center, and a Bt corn leaf and a conventional corn leaf (circular leaf) with a diameter of 1.5-2.5 cm (preferably 2.5 cm) are placed in each two adjacent units, and the Bt corn and conventional corn leaves are alternately and equidistantly placed along the periphery of the filter paper, and the distance from the center of each leaf to the center of the filter paper is equal; Preferably, the conventional corn is generally a Bt corn recipient variety with the same genetic background; (2) The pre-starved larvae of Lepidoptera insects are released in the center of the filter paper; (3) After the release of the larvae, the culture dish is sealed with plastic wrap to prevent the larvae from escaping, and 5-10 air holes are poked on the surface of the plastic wrap to ensure air circulation, and then the culture dish is covered with a dish cover and then covered with a black cloth to block light, and then transferred to an artificial climate chamber for breeding; (4) The positions and survival conditions of the larvae are observed and recorded at 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h after the release of the larvae, and the percentage of larvae feeding on different corn leaves is calculated, and the feeding selection rate is used to represent it; The calculation formula of the feeding selection rate is: (5) After the larvae feed for 48 h, the feeding area of the Bt corn and conventional corn leaves is recorded respectively, and the feeding selection index is calculated; each test is repeated 6-8 times (preferably 8 times).

[0010] The calculation formula of the feeding selection index is: Preferably, in step (1), the distance from the outer edge of the corn leaf to the edge of the filter paper is about 2 cm.

[0011] Further, in step (1), for 1-3 instar larvae, one corn leaf is placed in each unit; for 4 instar and above larvae, two overlapping corn leaves are placed in each unit.

[0012] Further, in step (2), for 1-3 instar larvae, 20 are introduced per dish; for 4 instar and above larvae, 5 are introduced per dish.

[0013] Further, the method for detecting the feeding selectivity of Lepidoptera on transgenic Bt corn and conventional corn according to the present application further comprises an initial feeding selection test, and the test method comprises: 1) laying a wet filter paper with the same size as the bottom of a 10 cm diameter culture dish on the bottom of the dish, and placing two pieces of Bt corn and conventional corn leaves with a diameter of 2.5 cm symmetrically along the center of the filter paper; 2) releasing one pre-starved 1-5 instar larva in the center of the filter paper, and observing the initial feeding selection of the larva within 15-45 s, including selecting Bt corn or conventional corn leaves, or individuals without selection; 3) testing 100 larvae per instar, repeated 3 times; all larvae are observed under the conditions of temperature 26 ± 1℃, relative humidity 60% ± 10%, and photoperiod 16 h light / 8 h dark. Among them, the artificial light source (light intensity about 8000-10000 lx) is equipped in the insect rearing room, and the LED light is used for artificial light supplement.

[0014] The method for detecting the feeding selectivity of Lepidoptera on transgenic Bt corn and conventional corn according to the present application further comprises a single leaf feeding persistence test, and the test method comprises: a) laying a wet filter paper with the same size as the bottom of a 20 cm diameter culture dish on the bottom of the dish, and placing Bt corn leaves or conventional corn leaves (circular leaves) with a diameter of 1.5-2.5 cm (preferably a diameter of 2.5 cm) in eight units equally divided from the center of the filter paper, each unit placing corn leaves along the periphery of the filter paper alternately and equidistantly, and the center of each leaf to the center of the filter paper is equal; wherein, for 1-3 instar larvae, one piece of corn leaf is placed in each unit; for 4 instar and above, two overlapping corn leaves are placed in each unit; b) releasing pre-starved larvae of Lepidoptera in the center of the filter paper; wherein, for 1-3 instar larvae, 20 are introduced per dish; for 4 instar and above, 5 are introduced per dish; c) after releasing the larvae, the culture dish is sealed with plastic wrap to prevent the larvae from escaping; 5-10 air holes are poked on the surface of the plastic wrap to ensure air circulation; then the culture dish is covered with a dish cover, and then covered with a black cloth to block light, and then transferred to an artificial climate chamber for rearing; d) observing and recording the position and survival of the larvae at 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h after the release of the larvae, and calculating the percentage of larvae feeding on different corn leaves, which is expressed by the feeding selection rate; e) Measure the leaf area consumed by the larvae 48 h after feeding; repeat each test 6-8 times (preferably 8 times). For example, a structural adhesive transparent sheet (1 mm × 1 mm) can be used to measure the leaf area consumed by the larvae.

[0015] Furthermore, the larvae are pre-starved for 6 hours.

[0016] Furthermore, the rearing conditions for the larvae were: a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h light / 8 h darkness.

[0017] Furthermore, the corn leaves are from corn leaves grown to the V5-V6 stage.

[0018] Secondly, the present invention provides a method for detecting the oviposition selectivity of lepidopteran insects to transgenic Bt maize, comprising: S1. Set up the following six processing groups ①~⑥ respectively: ① Healthy Bt corn only; ② Healthy conventional corn only; ③ Healthy Bt corn and healthy conventional corn; ④ Healthy Bt corn and damaged conventional corn; ⑤ Damaged Bt corn and healthy conventional corn; ⑥ Damaged Bt corn and damaged conventional corn. The preparation of the affected Bt corn and the affected conventional corn is as follows: When the corn grows to the V4 stage, 8-15 (preferably 8) newly hatched lepidopteran insect larvae are artificially introduced into the core leaf of each pot of Bt corn or conventional corn plant, and they are allowed to feed and cause damage for 48 hours; 2 corn seedlings are planted in each pot; during the experiment, the larvae are always kept on the corn plant to maintain a continuous feeding state. S2. Release 8-12 pairs (preferably 10 pairs) of newly emerged adult lepidopteran insects, with a male-to-female ratio of 1:1, into the insect rearing cage. S3. One day after the adults mate, place 6 pots of corn in the cage; For treatment groups ① and ②, 6 pots of corn were placed at equal intervals in the cage; for treatment groups ③ to ⑥, 3 pots of Bt corn and 3 pots of conventional corn, for a total of 6 pots of corn, were placed at equal intervals alternately in the cage. S4. During the oviposition period, replace oviposition-affected corn plants with fresh corn plants that have not been affected by oviposition every day; record the number of oviposition masses and the total number of oviposition grains on each type of corn for 5-7 consecutive days, and evaluate the oviposition status of Bt corn and conventional corn on the last day of the survey.

[0019] Preferably, a plastic culture dish containing cotton balls soaked in 10% honey water is placed in the center of the insect rearing cage to supplement the adult insects with nutrients and water.

[0020] Preferably, the insect cage has dimensions of 60 cm × 60 cm × 60 cm and a 120-mesh screen; and / or, Furthermore, the experiment was conducted at a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h light / 8 h darkness; each experiment was repeated 4-6 times (preferably 4 times).

[0021] Release them into a 120-mesh insect rearing cage (60 cm × 60 cm × 60 cm).

[0022] In this invention, the lepidopteran insects are selected from beet armyworm, Asian corn borer, or armyworm, etc.; beet armyworm is preferred.

[0023] In this invention, Bt corn is a corn variety that expresses one or more Bt insecticidal proteins (such as Bt corn Huaxingdan 88-DBN3601T), and conventional corn is the recipient variety of this Bt corn (such as 'Huaxingdan 88').

[0024] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention systematically studies the feeding and oviposition selectivity of the beet armyworm towards Bt and conventional maize, clarifying the feeding selectivity of beet armyworm larvae in the initial stage, the feeding inhibition effect of Bt maize on beet armyworm larvae, and the differences in oviposition preferences of adults between infested and uninfested maize plants. Compared with existing technologies, this invention has significant innovations and advantages in terms of test materials, experimental design, feeding selectivity assessment methods, and oviposition behavior observation, mainly reflected in the following aspects: (i) The selection of test materials is innovative. This invention can selectively test the feeding and oviposition behavior of larvae between transgenic Bt maize and conventional recipient maize, revealing changes in pest avoidance responses before contact with plant tissues, indirectly assessing the differences in metabolites between transgenic Bt maize plants and conventional maize plants, and the potential changes in volatile metabolites after maize plants are damaged. It can meet the needs of assessing the effects of transgenic Bt maize metabolites and the needs of ecological and environmental safety evaluation.

[0025] (ii) The experimental design of selective larval feeding is innovative. This invention uses beet armyworm larvae of different instars (1st to 5th instar) to observe their feeding behavior on Bt corn and conventional corn leaves in a controlled environment, and records in detail the feeding location, feeding area, and larval survival at different time points. This method avoids interference from external environmental factors (such as light and air circulation), making the experimental conditions more controllable and reproducible.

[0026] Compared to traditional feeding selection experiments, this invention has significant advantages in the following aspects: 1. Precise control of larval starvation time: By starving the larvae for 6 hours before the experiment, the random feeding behavior of the larvae was effectively reduced, making their selective behavior more representative. 2. Dynamic observation of feeding behavior: By setting multiple time points (1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h), the feeding location and survival status of larvae are continuously observed. Compared with the traditional single survey time, this method reveals the dynamic changes in the feeding behavior of larvae more comprehensively. 3. Precisely quantify feeding area: The area of ​​leaf damage is measured using a structural adhesive transparent film (1 mm × 1 mm), making feeding data more intuitive and quantifiable, and avoiding the subjective errors of traditional visual recording.

[0027] The experimental results showed that, under the same conditions, beet armyworm larvae from the 1st to 5th instar exhibited significant feeding refusal and avoidance behaviors, namely, they preferred to feed on conventional corn leaves, while their consumption of Bt corn leaves was significantly reduced, especially in the 1st to 3rd instar larvae. These results further confirm the significant inhibitory effect of Bt corn on beet armyworm larvae feeding and provide important experimental evidence for field pest control.

[0028] (III) Improvements to the experimental design of oviposition selection This invention, in an experiment on adult oviposition selectivity, simulated the differences in plant growth induced by pests between Bt and conventional corn under natural field conditions by setting up combined treatments of healthy and infested plants. This accurately reflects the oviposition selection behavior of female beet armyworm adults. Compared with existing research, this invention has the following advantages in experimental design and data acquisition: 1. Simulate field pest stress environment Traditional experiments often use healthy plants to study oviposition selectivity. However, this invention sets up multiple treatment groups, such as undamaged Bt corn and undamaged conventional corn, undamaged Bt corn and damaged conventional corn, damaged Bt corn and undamaged conventional corn, and damaged Bt corn and damaged conventional corn, which can comprehensively simulate different pest scenarios in the field and provide a more realistic reference for the oviposition selectivity of beet armyworm adults.

[0029] 2. Dynamically record the number and location of eggs laid. This invention ensures the accuracy and systematic nature of data acquisition by replacing oviposition plants daily, recording the number and distribution of eggs, and simultaneously monitoring the damage level of plants five days after oviposition. Compared to traditional experiments that only record oviposition data at a single time point, this data recording method can more comprehensively reflect the changing trends of beet armyworm oviposition behavior and improve the reliability of research results.

[0030] 3. The experimental method is simple and efficient, and suitable for large-scale application. This invention simplifies and streamlines experimental procedures and equipment selection, making the experimental methods easier to operate and offering the following advantages: (1) Experimental equipment is readily available: The experiment uses conventional experimental equipment such as glass petri dishes, qualitative filter paper, plastic wrap, and hole punch, and the equipment is inexpensive and easy to operate, making it easy to promote and apply on a large scale. (2) Fast and accurate data acquisition: The structural adhesive transparent film is used to accurately measure the damaged area of ​​the leaves, replacing the traditional visual assessment method, which greatly improves the accuracy of the data and reduces human error; (3) Controllable experimental cycle: By controlling the experimental time within 48 hours (larvae) and within 5 days (adults), the experimental cycle is significantly shortened and the experimental efficiency is improved, providing a data basis for further large-scale field verification experiments. Attached Figure Description

[0031] Figure 1 This is a preferred embodiment of the beet armyworm larvae's attraction to Bt corn and conventional corn. A shows the overall structure of the larval attraction selection device; B shows the arrangement of Bt corn and conventional corn leaves in the device.

[0032] Figure 2 This invention provides a preferred embodiment of a device for determining the feeding selectivity of beet armyworm larvae towards Bt corn and conventional corn. A represents the overall structure of the larval feeding selectivity determination device; B and C represent the arrangement of Bt corn and conventional corn leaves in the device under the same type of leaf supply conditions; D represents the arrangement of Bt corn and conventional corn leaves in the device under alternating leaf supply conditions.

[0033] Figure 3 This invention provides a preferred embodiment of a device for determining the selectivity of adult beet armyworms in oviposition on Bt corn and conventional corn. Detailed Implementation

[0034] This invention aims to provide a simple and standardized method for detecting the feeding preferences of larvae and oviposition preferences of lepidopteran pests (such as the beet armyworm, Asian corn borer, and armyworm) between Bt maize and conventional maize, thereby solving the problems of cumbersome operation and discontinuous data recording in existing methods. This method can rapidly assess the impact of Bt maize on the behavior of target pests, indirectly reflecting the differences in volatile compounds and secondary metabolites in Bt maize, providing a scientific basis for the environmental safety assessment, optimization of resistance management strategies, and sustainable development of Bt maize. Furthermore, combining qualitative and quantitative analysis of volatile compounds and secondary metabolites in maize plants helps to further explore the intrinsic mechanisms of insect behavioral selection.

[0035] This invention provides a method for detecting the feeding and oviposition selectivity of lepidopteran insects to transgenic Bt maize, comprising the following technical solutions: 1. Test insect source The beet armyworm was used as the research subject. Larvae were collected and placed in a controlled environment with a temperature of 26 ± 1℃, relative humidity of 60% ± 10%, and a photoperiod of 16 h / 8 h (L / D), and were fed an artificial diet based on wheat bran and soybean meal. Adults (female:male = 1:1) were reared in plastic boxes with a diameter of 12 cm and a height of 14 cm, covered with sterile gauze, and fed 10% honey water (such as acacia honey or other suitable honey) to supplement nutrition and hydration. Gauze containing egg masses was collected daily and transferred to 6 cm × 8 cm resealable bags for incubation.

[0036] 2. Test plants Transgenic Bt maize (transformation event expressing Bt insecticidal protein, expressing or not expressing herbicide-resistant genes) was used, while the control group consisted of conventional hybrid maize varieties with the same genetic background. Maize was grown in a greenhouse at a temperature of 28 ± 1℃, relative humidity of 80% ± 5%, and natural photoperiod. Three seeds were sown per plastic pot (9 cm in diameter, 10 cm in height) containing a soil-substrate mixture, and two healthy seedlings were retained after germination.

[0037] 3. Test equipment Glass petri dishes (10 cm in diameter), glass petri dishes (20 cm in diameter), qualitative filter paper (10 cm in diameter), qualitative filter paper (20 cm in diameter), hole punch (2.5 cm in diameter), plastic wrap, structural adhesive transparent film (1 mm × 1 mm) (purchased from Beijing Zhongke Luda Test Instrument Co., Ltd.), 120 mesh insect cage (60 cm × 60 cm × 60 cm).

[0038] 4. Test methods 4.1 Feeding selectivity of beet armyworm larvae on Bt and conventional maize When the corn grown in the greenhouse reaches the V5-V6 stage, the newest fully expanded leaves are cut off, and circular leaves are obtained using a 2.5 cm diameter circular punch. These leaves are then used to determine the feeding selectivity under the following three different experimental conditions.

[0039] (1) Initial feeding selection test: Place a moistened filter paper at the bottom of a 10 cm diameter petri dish, and symmetrically place two Bt corn leaves (2.5 cm in diameter) and a regular corn leaf along the center of the filter paper.

[0040] Release one 1st-5th instar larva that has been pre-starved for 6 hours into the center of the filter paper and observe the larvae's initial selection within 30 seconds, including selecting Bt corn or regular corn leaves, or individuals that do not make a selection.

[0041] One hundred larvae were tested at each instar, with three replicates. All larvae were observed in a controlled environment with a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h / 8 h (L / D).

[0042] (2) Single leaf feeding persistence test: Place a moistened filter paper at the bottom of a 20 cm diameter petri dish, and place 8 leaves of the same type of corn (Bt corn or conventional corn) with a diameter of 2.5 cm at equal intervals around the filter paper (for 5th instar larvae, use two leaves overlapping each other, for a total of 16 leaves).

[0043] Release 1st to 5th instar larvae that have been starved for 6 hours into the center of the filter paper (20 1st to 3rd instar larvae per dish; 5 4th to 5th instar larvae per dish).

[0044] After the larvae are released, they are covered with double-layered black cotton cloth to block out the light and then transferred to an artificial climate chamber with a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h / 8 h (L / D) for rearing.

[0045] The location and survival status of the larvae were observed and recorded at 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h after larval release. The percentage of larvae feeding on the leaves of different maize varieties was calculated and expressed as the feeding selection rate.

[0046] Forty-eight hours after feeding, the leaf area consumed by the larvae was measured using a structural adhesive transparent sheet (1 mm × 1 mm). Each test was repeated eight times.

[0047] (3) Selective feeding experiment of mixed leaves: Place a moistened filter paper at the bottom of a 20 cm diameter petri dish, and alternately place 8 Bt corn and regular corn leaves with a diameter of 2.5 cm at equal intervals around the filter paper (for 5th instar larvae, use two overlapping leaves, for a total of 16 leaves).

[0048] Release 1st to 5th instar larvae that have been starved for 6 hours into the center of the filter paper (20 1st to 3rd instar larvae per dish; 5 4th to 5th instar larvae per dish).

[0049] After the larvae are released, they are covered with double-layered black cotton cloth to block out the light and then transferred to an artificial climate chamber with a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h / 8 h (L / D) for rearing.

[0050] The location and survival status of the larvae were observed and recorded at 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h after larval release. The percentage of larvae feeding on the leaves of different maize varieties was calculated and expressed as the feeding selection rate.

[0051] Forty-eight hours after feeding, the grazing area of ​​leaves from Bt maize and conventional maize was recorded, and the grazing selection index was calculated. Each test was repeated eight times.

[0052] 4.2 Oviposition selectivity of adult beet armyworms on Bt and conventional maize The following four types of corn plants were prepared in advance: (A) healthy Bt corn; (B) healthy conventional corn; (C) Bt corn damaged by larvae; (D) conventional corn damaged by larvae.

[0053] The larval damage was treated as follows: Eight newly hatched beet armyworm larvae were artificially introduced into the core leaf of each Bt or conventional corn plant and allowed to feed continuously for 48 hours. All corn plants were from the aforementioned greenhouse-grown corn, at stage V4, with two seedlings per plant. Throughout the experiment, the larvae remained on the plant to maintain continuous feeding.

[0054] The experiment was set up with 6 treatments: (a) Bt corn only; (b) conventional corn only; (c) undamaged Bt corn and undamaged conventional corn; (d) undamaged Bt corn and damaged conventional corn; (e) damaged Bt corn and undamaged conventional corn; (f) damaged Bt corn and damaged conventional corn.

[0055] In each experimental combination, 10 pairs of newly emerged beet armyworm adults (female:male = 1:1) were released into a 120-mesh rearing cage (60 cm × 60 cm × 60 cm).

[0056] A plastic petri dish containing cotton balls soaked in 10% honey water was placed in the center of the cage to supplement the adult insects with nutrients and water.

[0057] One day after the adults mate, six pots of corn (three pots of Bt corn and three pots of regular corn) are placed alternately at equal intervals in the cage.

[0058] During the oviposition period, oviposition-affected plants were replaced daily with fresh, unaffected plants. The number of oviposition masses and the total number of oviposition kernels on each type of maize were recorded for 5 consecutive days, and the damage to Bt and conventional maize was assessed on day 5.

[0059] The experiment was conducted in a greenhouse with a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h / 8 h (L / D). Each experiment was repeated 4 times.

[0060] The experiments of this invention found that beet armyworm larvae from the 1st to 5th instar prefer to feed on conventional corn leaves, and all of them show a refusal to feed and avoidance behavior on Bt corn leaves, and this behavior is more pronounced in the younger larvae.

[0061] Beet armyworm adults do not show a clear oviposition preference between healthy Bt and conventional corn plants. However, when conventional corn is infested by the pest, female moths tend to lay their eggs on healthy or undamaged Bt corn plants.

[0062] The key technical points of this invention include at least: 1. Larval selection and starvation treatment: Randomly select larvae in the pre-molting state and starve them for 6 hours after they molt and enter the next instar. 2. Number of leaf discs: Adjust the number of leaf discs according to the feeding needs of larvae at different instars; 3. Maintaining humidity in the petri dish: Place moist filter paper at the bottom of the petri dish to maintain the freshness of the leaves and prevent wilting from affecting the larvae's feeding behavior; 4. Sealing and air permeability of petri dishes: Seal the lid with plastic wrap to prevent larvae from escaping; use a fine needle to poke 5-10 air holes on the surface of the plastic wrap to ensure air circulation; 5. Avoid light interference: The experiment should be conducted under shaded conditions to prevent the larvae from affecting their feeding selection due to phototaxis; 6. Measurement of feeding area: 48 h after the larvae have been fed, the affected area of ​​the leaves was measured using a transparent sheet of structural adhesive (1 mm × 1 mm grid). 7. Adult selection: Select healthy, normal-sized adults that have emerged within 24 hours (such as beet armyworm, Asian corn borer, or armyworm).

[0063] 8. Mating and pairing: Pairing is carried out according to a female:male ratio of 1:1, with 10 pairs of adult insects released in each rearing cage to ensure a high success rate of mating; 9. Replacement of egg-laying plants: Replace all corn plants that have laid eggs daily to prevent the release of chemical signals from the egg masses from interfering with subsequent egg-laying behavior; 10. Nutritional supplementation for adults: Place cotton balls containing 10% honey water in the rearing cage to ensure that the adults receive sufficient nutrition and improve their egg-laying vitality; 11. Assessment of larval feeding damage: On the 5th day after the adult lays eggs, record the degree of feeding damage on the corn plants, including the affected area and the level of damage. 12. Removal of egg masses from the mesh screen: Clean the egg masses covering the mesh screen daily to prevent hatching larvae from interfering with the experiment; 13. Environmental conditions control: Temperature, humidity and light conditions must be strictly controlled during the experiment to ensure air circulation in the breeding room.

[0064] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0065] Example 1: Feeding selectivity of beet armyworm larvae on Bt and conventional corn 1. Test insect source The beet armyworm was used as the research subject. Larvae were collected and placed in a controlled environment with a temperature of 26 ± 1℃, relative humidity of 60% ± 10%, and a photoperiod of 16 h / 8 h (L / D), and fed an artificial diet based on wheat bran and soybean meal (mass ratio 15:8). Emerging adults (female:male = 1:1) were reared in plastic boxes with a diameter of 12 cm and a height of 14 cm, covered with sterile gauze, and fed 10% honey water to supplement nutrition and hydration. Gauze containing egg masses was collected daily and transferred to 6 cm × 8 cm resealable bags for incubation.

[0066] 2. Test plants The transformation event for transgenic Bt maize was DBN3601T (expressing Cry1Ab and Vip3Aa Bt insecticidal proteins and Epsps and Pat genes). The control group was the conventional hybrid maize variety 'Huaxingdan 88' with the same genetic background. Seeds were provided by Beijing Dabeinong Biotechnology Co., Ltd. Maize was grown in a greenhouse at a temperature of 28 ± 1℃, a relative humidity of 80% ± 5%, and a natural photoperiod. Three seeds were sown per pot in plastic pots (9 cm in diameter, 10 cm in height) containing a soil-substrate mixture, and two healthy seedlings were retained after germination.

[0067] 3. Test equipment Glass petri dish (10 cm in diameter), glass petri dish (20 cm in diameter), qualitative filter paper (10 cm in diameter), qualitative filter paper (20 cm in diameter), hole punch (2.5 cm in diameter), plastic wrap, structural adhesive transparent film (1 mm × 1 mm).

[0068] 4. Test methods When the corn grown in the greenhouse reaches the V5-V6 stage, the newest fully expanded leaves are cut off, and circular leaves are obtained using a 2.5 cm diameter circular punch. These leaves are then used to determine the feeding selectivity under the following three different experimental conditions.

[0069] (1) Initial feeding selection test: Place a moistened filter paper at the bottom of a 10 cm diameter petri dish, and symmetrically place two Bt corn leaves (2.5 cm in diameter) and a regular corn leaf along the center of the filter paper.

[0070] Release one 1st-5th instar larva that has been pre-starved for 6 hours into the center of the filter paper and observe the larvae's initial selection within 30 seconds, including selecting Bt corn or regular corn leaves, or individuals that do not make a selection.

[0071] One hundred larvae were tested at each instar, with three replicates. All larvae were observed in a controlled environment with a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h / 8 h (L / D).

[0072] The beet armyworm larvae's tropism selection device for Bt and conventional maize is shown in [link to relevant documentation]. Figure 1 .

[0073] (2) Single leaf feeding persistence test: Place a moistened filter paper at the bottom of a 20 cm diameter petri dish, and place 8 leaves of the same type of corn (Bt corn or conventional corn) with a diameter of 2.5 cm at equal intervals around the filter paper (for 5th instar larvae, use two leaves overlapping each other, for a total of 16 leaves).

[0074] Release 1st to 5th instar larvae that have been starved for 6 hours into the center of the filter paper (20 1st to 3rd instar larvae per dish; 5 4th to 5th instar larvae per dish).

[0075] After the larvae are released, they are covered with double-layered black cotton cloth to block out the light and then transferred to an artificial climate chamber with a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h / 8 h (L / D) for rearing.

[0076] The location and survival status of the larvae were observed and recorded at 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h after larval release. The percentage of larvae feeding on the leaves of different maize varieties was calculated and expressed as the feeding selection rate.

[0077] Forty-eight hours after feeding, the leaf area consumed by the larvae was measured using a structural adhesive transparent sheet (1 mm × 1 mm). Each test was repeated eight times.

[0078] (3) Selective feeding experiment of mixed leaves: Place a moistened filter paper at the bottom of a 20 cm diameter petri dish. Alternately place eight Bt corn and regular corn leaves (2.5 cm in diameter) at equal intervals around the filter paper (for 5th instar larvae, use two overlapping leaves, for a total of 16 leaves) around the filter paper. The distance from the outer edge of the corn leaf to the edge of the filter paper is approximately 2 cm.

[0079] Release 1st to 5th instar larvae that have been starved for 6 hours into the center of the filter paper (20 1st to 3rd instar larvae per dish; 5 4th to 5th instar larvae per dish).

[0080] After the larvae are released, they are covered with double-layered black cotton cloth to block out the light and then transferred to an artificial climate chamber with a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h / 8 h (L / D) for rearing.

[0081] The location and survival status of the larvae were observed and recorded at 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h after larval release. The percentage of larvae feeding on the leaves of different maize varieties was calculated and expressed as the feeding selection rate.

[0082] Forty-eight hours after feeding, the grazing area of ​​leaves from Bt maize and conventional maize was recorded, and the grazing selection index was calculated. Each test was repeated eight times.

[0083] The apparatus for measuring the feeding selectivity of beet armyworm larvae on Bt and conventional maize is shown in [link to device]. Figure 2 .

[0084] 5. Results The tropism response of beet armyworm larvae to Bt and conventional maize showed that 1st-5th instar larvae did not exhibit significant differences in preference between Bt and conventional maize leaf tissues upon initial selection. Bt maize did not have a significant attraction or repulsion effect on 1st-5th instar beet armyworm larvae. This indicates that Bt gene transfection did not significantly alter the insect's feeding selection behavior, indirectly suggesting that it did not cause significant changes in the larvae's tropism-related volatile chemical substances.

[0085] Under the same leaf feeding conditions, the feeding selection rates of 1st, 2nd, 3rd, and 5th instar larvae on Bt maize were significantly lower than those on conventional maize. As feeding time progressed, the feeding selection rates of 1st-5th instar larvae on Bt maize gradually decreased, while those on conventional maize gradually increased. After 48 hours, the feeding selection rates of 1st, 2nd, 3rd, and 4th instar larvae on Bt maize were 8.34%, 30.63%, 52.26%, and 76.89%, respectively, all lower than those on conventional maize (67.88%, 76.88%, 86.88%, and 96.88%, respectively). The feeding selection rates of 5th instar larvae on Bt maize and conventional maize were 90.00% and 100.00%, respectively, with no significant difference. After feeding 1st-5th instar larvae with leaves for 48 hours, the larvae's feeding area on Bt maize leaves was significantly lower than that on conventional maize. Among them, the feeding area of ​​3rd instar larvae on conventional corn and Bt corn differed by 166.92 times.

[0086] Under alternating feeding conditions with two types of maize leaves, the selection rates of 1st-5th instar larvae of the beet armyworm on Bt maize were significantly lower than those on conventional maize. As feeding time progressed, the selection rates of 1st, 2nd, and 5th instar larvae on Bt maize gradually decreased, while they gradually increased on conventional maize. For 3rd instar larvae, the selection rate on Bt maize initially decreased, then increased, and then decreased again, before slowly increasing on conventional maize. For 4th instar larvae, the selection rate on Bt maize decreased slowly, while on conventional maize it initially decreased and then increased. After 48 hours of continuous feeding, the selection rates of 1st, 2nd, 3rd, and 4th instar larvae on Bt maize were 9.38%, 18.75%, 22.50%, and 33.75%, respectively, significantly lower than their selection rates on conventional maize (48.32%, 66.25%, 40.63%, and 50.00%, respectively). The selection rates of 5th instar larvae on Bt maize and non-Bt maize were 33.75% and 56.25%, respectively. After feeding beet armyworm larvae from the first to fifth instars with both Bt corn and conventional corn for 48 hours, the feeding area and feeding selection index of the first to fifth instar larvae on Bt corn leaves were significantly lower than those on conventional corn.

[0087] Example 2: Oviposition preferences of beet armyworm adults for Bt and conventional corn 1. Test insect source The beet armyworm was used as the research subject. Larvae were collected from fresh corn fields and placed in a controlled environment with a temperature of 26 ± 1℃, relative humidity of 60% ± 10%, and a photoperiod of 16 h / 8 h (L / D), fed with an artificial diet based on wheat bran and soybean meal. Emerging adults (female:male = 1:1) were reared in plastic boxes with a diameter of 12 cm and a height of 14 cm, covered with sterile gauze, and fed 10% honey water to supplement nutrition and moisture. Gauze containing egg masses was collected daily and transferred to 6 cm × 8 cm resealable bags for incubation.

[0088] 2. Test plants The transformation event for transgenic Bt maize was DBN3601T (expressing Cry1Ab and Vip3Aa Bt insecticidal proteins and Epsps and Pat genes). The control group was the conventional hybrid maize variety 'Huaxingdan 88' with the same genetic background. Seeds were provided by Beijing Dabeinong Biotechnology Co., Ltd. Maize was grown in a greenhouse at a temperature of 28 ± 1℃, a relative humidity of 80% ± 5%, and a natural photoperiod. Three seeds were sown per pot in plastic pots (9 cm in diameter, 10 cm in height) containing a soil-substrate mixture, and two healthy seedlings were retained after germination.

[0089] 3. Test equipment 120-mesh insect rearing cage (60 cm × 60 cm × 60 cm).

[0090] 4. Test methods Prepare the following four types of corn plants in advance: (A) healthy Bt corn; (B) healthy conventional corn; (C) Bt corn damaged by larvae; (D) conventional corn damaged by larvae.

[0091] The larval damage was treated as follows: Eight newly hatched beet armyworm larvae were artificially introduced into the core leaf of each Bt or conventional corn plant and allowed to feed continuously for 48 hours. All corn plants were from the aforementioned greenhouse-grown corn, at stage V4, with two seedlings per plant. Throughout the experiment, the larvae remained on the plant to maintain continuous feeding.

[0092] The experiment was set up with 6 treatments: (a) Bt corn only; (b) conventional corn only; (c) undamaged Bt corn and undamaged conventional corn; (d) undamaged Bt corn and damaged conventional corn; (e) damaged Bt corn and undamaged conventional corn; (f) damaged Bt corn and damaged conventional corn.

[0093] In each experimental combination, 10 pairs of newly emerged beet armyworm adults (female:male = 1:1) were released into a 120-mesh rearing cage (60 cm × 60 cm × 60 cm).

[0094] A plastic petri dish containing cotton balls soaked in 10% honey water was placed in the center of the cage to supplement the adult insects with nutrients and water.

[0095] One day after the adults mate, six pots of corn (three pots of Bt corn and three pots of regular corn) are placed alternately at equal intervals in the cage.

[0096] During the oviposition period, oviposition-affected plants were replaced daily with fresh, unaffected plants. The number of oviposition masses and the total number of oviposition kernels on each type of maize were recorded for 5 consecutive days, and the damage to Bt and conventional maize was assessed on day 5.

[0097] The experiment was conducted in a greenhouse with a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h / 8 h (L / D). Each experiment was repeated 4 times.

[0098] The apparatus for determining the oviposition selectivity of beet armyworm adults to Bt and conventional maize is shown in [link to device]. Figure 3 .

[0099] 5. Results When only Bt or conventional corn plants were present, the total number of egg masses and the number of eggs laid by adult beet armyworms on Bt corn plants were 7.25 and 430.00 respectively (average of 4 replicates), while on conventional corn plants, the numbers were 7.50 and 343.50 respectively (average of 4 replicates). There was no significant difference in egg production between Bt and conventional corn plants. This indicates that the beet armyworm did not exhibit significant tropism towards the plant volatiles of either Bt or conventional corn plants, indirectly suggesting that this transgenic transformation event did not lead to significant changes in the insect's tropistic metabolic volatiles.

[0100] Without considering plant damage or the presence of the same larvae, there was no significant difference in egg production between adult beet armyworms on healthy Bt corn and conventional corn. Considering the different degrees of damage to Bt and conventional corn plants in the field, we treated both with newly hatched larvae. The results showed that the damage to Bt corn was significantly lower than that to conventional corn. When Bt and conventional corn plants with different levels of damage coexisted, the beet armyworm laid more eggs on healthy Bt corn (73.55% of total eggs) or less damaged Bt corn (66.67% of total eggs). Significant differences in egg production were observed between healthy Bt corn and damaged conventional corn, and between damaged Bt corn and damaged conventional corn. The total number of eggs laid by adults on healthy Bt corn and damaged Bt corn was 2.78 times and 2.00 times that on damaged non-Bt corn plants, respectively. However, there was no significant difference in egg production between damaged Bt corn and healthy conventional corn. The results revealed that after beet armyworm larvae feed on corn plants, they can induce changes in the volatile compounds released by the plants, causing adults to prefer laying eggs on relatively healthy Bt plants. The potential impact of this behavior should be fully considered when implementing shelter strategies. On the other hand, the "death trap" effect of Bt corn plants can be used to attract adults to lay eggs, and the hatched larvae are killed by Bt proteins, thereby reducing pest stress.

[0101] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting the feeding selectivity of lepidopteran insects towards transgenic Bt maize and conventional maize, characterized in that, Includes the following steps: (1) Lay a moist filter paper of the same size as the bottom of the petri dish with a diameter of 10-20 cm. Divide the filter paper into eight equal units with the center of the filter paper as the center. Place Bt corn leaves and regular corn leaves with a diameter of 1.5-2.5 cm in each two adjacent units. Place Bt corn and regular corn leaves alternately and equidistantly around the filter paper, and the distance from the center of each leaf to the center of the filter paper is equal. Preferably, the conventional maize is a Bt maize recipient variety with the same genetic background; (2) Release pre-starved lepidopteran larvae into the center of the filter paper; (3) After the larvae are released, seal the petri dish with plastic wrap to prevent the larvae from escaping; Make 5-10 ventilation holes in the plastic wrap to ensure air circulation; then cover the petri dish with a lid, then cover it with a black cloth to block out the light, and transfer it to an artificial climate chamber for rearing. (4) Observe and record the location and survival status of the larvae at 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h after larval release, and calculate the percentage of larvae feeding on different corn leaves, expressed as the feeding selection rate; the formula for calculating the feeding selection rate is: (5) After 48 hours of feeding by the larvae, the feeding area of ​​leaves of Bt corn and conventional corn was recorded respectively, and the feeding selection index was calculated; the formula for calculating the feeding selection index is: Each test group was repeated 6-8 times.

2. The method according to claim 1, characterized in that, Step (1) The distance from the outer edge of the corn leaf to the edge of the filter paper is 2 cm.

3. The method according to claim 1, characterized in that, Step (1) For 1st to 3rd instar larvae, place one corn leaf in each unit; for 4th instar and above larvae, place two overlapping corn leaves in each unit.

4. The method according to claim 1, characterized in that, Step (2) For 1st to 3rd instar larvae, 20 larvae are introduced per dish; for 4th instar and above larvae, 5 larvae are introduced per dish.

5. The method according to any one of claims 1-4, characterized in that, It also includes initial feeding selection tests, the methods of which include: 1) Place a moistened filter paper of the same size as the bottom of a 10 cm diameter petri dish at the bottom, and symmetrically place two Bt corn leaves and a regular corn leaf of 2.5 cm diameter along the center of the filter paper. 2) Release one pre-starved 1st-5th instar larva in the center of the filter paper and observe the larva's initial feeding choices within 15-45 seconds, including choosing Bt corn or regular corn leaves, or individuals that do not make a choice. 3) 100 larvae were tested for each instar, and the test was repeated 3 times. All larvae were observed under the conditions of a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h light / 8 h darkness.

6. The method according to claim 5, characterized in that, Further, it includes a single-leaf feeding persistence test, the test methods of which include: a) Place a moistened filter paper of the same size as the bottom of a 20 cm diameter petri dish at the bottom. Divide the filter paper into eight equal units with the center of the filter paper as the center. Place Bt corn or regular corn leaves with a diameter of 1.5-2.5 cm in each unit. Place the corn leaves alternately and equidistantly around the filter paper, and the distance from the center of each leaf to the center of the filter paper is equal. For 1st to 3rd instar larvae, one corn leaf is placed in each unit; for 4th instar and above larvae, two overlapping corn leaves are placed in each unit. b) Release pre-starved lepidopteran larvae into the center of the filter paper; For 1st to 3rd instar larvae, 20 larvae are introduced per dish; for 4th instar and above larvae, 5 larvae are introduced per dish. c) After the larvae are released, seal the petri dish with plastic wrap to prevent the larvae from escaping; poke 5-10 ventilation holes in the plastic wrap with a needle to ensure air circulation; then cover the petri dish with a lid, then cover it with a black cloth to block out the light, and transfer it to an artificial climate chamber for rearing. d) Observe and record the location and survival status of the larvae at 1, 2, 4, 6, 8, 10, 12, 24, 36 and 48 h after larval release, and calculate the percentage of larvae feeding on different maize leaves, expressed as feeding selection rate; e) Measure the leaf area fed by the larvae 48 h after feeding; repeat each test 6-8 times.

7. The method according to any one of claims 1-6, characterized in that, The larvae are pre-starved for 6 hours; and / or, The rearing conditions for larvae are: temperature 26 ± 1℃, relative humidity 60% ± 10%, and photoperiod of 16 h light / 8 h darkness; and / or, The corn leaves are from the corn leaves that have grown to the V5-V6 stage.

8. A method for detecting the oviposition selectivity of lepidopteran insects towards transgenic Bt maize and conventional maize, characterized in that, include: S1. Set up the following six processing groups ①~⑥ respectively: ① Healthy Bt corn only; ② Healthy conventional corn only; ③ Healthy Bt corn and healthy conventional corn; ④ Healthy Bt corn and damaged conventional corn; ⑤ Damaged Bt corn and healthy conventional corn; ⑥ Damaged Bt corn and damaged conventional corn. The preparation of the affected Bt corn and the affected conventional corn is as follows: When the corn grows to the V4 stage, 8-15 newly hatched lepidopteran larvae are artificially introduced into the core leaf of each Bt corn or conventional corn plant and allowed to feed and cause damage for 48 hours; 2 corn seedlings are planted in each pot; during the experiment, the larvae are always kept on the corn plant to maintain a continuous feeding state. S2. Release 8-12 pairs of newly emerged Lepidoptera adults, with a male-to-female ratio of 1:1, into the insect rearing cage. S3. One day after the adults mate, place 6 pots of corn in the cage; For treatment groups ① and ②, 6 pots of corn were placed at equal intervals in the cage; for treatment groups ③ to ⑥, 3 pots of Bt corn and 3 pots of conventional corn, for a total of 6 pots of corn, were placed at equal intervals alternately in the cage. S4. During the oviposition period, replace oviposition-affected corn plants with fresh corn plants that have not been affected by oviposition every day; record the number of oviposition masses and the total number of oviposition grains on each type of corn for 5-7 consecutive days, and evaluate the oviposition status of Bt corn and conventional corn on the last day of the survey.

9. The method according to claim 8, characterized in that, A plastic petri dish containing cotton balls soaked in a 10% honey solution is placed in the center of the insect rearing cage to provide nutrients and moisture for the adult insects; and / or, The insect cage measures 60 cm × 60 cm × 60 cm and has a 120-mesh screen; and / or, The experiment was conducted at a temperature of 26 ± 1℃, a relative humidity of 60% ± 10%, and a photoperiod of 16 h light / 8 h darkness; each experiment was repeated 4-6 times.

10. The method according to any one of claims 1-9, characterized in that, The lepidopteran insects are selected from beet armyworm, Asian corn borer, or armyworm; beet armyworm is preferred.

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