A method for rearing a fruit fly with a specialized diet dependent on a specific natural host plant

Abstract

The present application relates to insect rearing technology, and discloses a method for rearing fruit flies with specialized diet depending on specific natural host plants. The host plants are treated at low temperature, then restored to room temperature, cut into segments, and mixed to serve as food and oviposition substrate for larvae. Wet filter paper is laid in a culture tube, host plants, apples and kiwifruits are added, and fertilized female adults or male and female adults are introduced. The culture is carried out at 22 DEG C, 75% relative humidity, and 12 hours of light per day. Mouldy fruits are replaced regularly, mould spots and water droplets on the tube wall are cleaned, and oviposition, hatching, pupation and emergence are observed. The offspring adults are paired and the culture is continued. The method is easy to operate, and can realize continuous maintenance without the need of preparing artificial culture medium.

Description

Technical Field

[0001] This invention relates to the field of laboratory insect rearing technology, specifically to a method for rearing specialized fruit flies that rely on specific natural host plants, and more particularly to a method that uses treated natural host plants as oviposition substrates and larval food, supplemented by fruit pieces as adult food, and can maintain the target fruit flies for continuous generations under laboratory conditions. Background Technology

[0002] Fruit flies are commonly used laboratory insects in biological research. Some fruit flies are closely related to the classic model insect, the black-bellied fruit fly (Drosophila melanogaster), and have high application value in studies of genetics, development, reproduction, physiology, and behavior. With the development of related research, the demand for stable, continuous, and sufficient quantities of fruit fly experimental materials is constantly increasing. Therefore, establishing a laboratory rearing system suitable for the biological characteristics of the target fruit flies is crucial.

[0003] Different fruit flies have different diets and oviposition habits. Conventional laboratory fruit flies can grow and develop using general or artificially prepared culture media. However, some fruit flies have highly specialized diets. Their female adults are selective about oviposition sites, while their larvae rely on the tissues of specific natural host plants to obtain nutrition and complete their growth and development. If they are separated from their suitable host plants, problems may occur such as female adults not laying eggs, reduced egg hatching rate, larvae not being able to feed normally, insufficient larval vitality, inability to pupate normally, or a small number of larvae emerging as adults.

[0004] The target fruit fly is a fruit fly with a highly specialized diet that is difficult to stably raise under conventional conditions. Through long-term observation of its wild living environment, adult activity sites, oviposition locations, and larval feeding objects, it was found that *Hymenochloa chinensis* is an important natural host plant for this target fruit fly. The tender leaves and stems of *Hymenochloa chinensis* can serve as food for the larvae of the target fruit fly and provide a suitable oviposition site for the target fruit fly. Adult target fruit flies will also occasionally lick *Hymenochloa chinensis*.

[0005] If the adult flies are fed only with general food without a suitable hyssop substrate during laboratory rearing, the target fruit flies may lack suitable oviposition sites, and the larvae may not be able to grow and develop normally due to the lack of specific host plants. Therefore, the rearing of this type of fruit fly cannot simply follow the culture medium rearing method of general fruit flies, but needs to meet the needs of different stages such as adult nutritional supplementation, female adult oviposition selection, and larval specialized feeding.

[0006] There are also some problems with directly placing wild-collected hyacinths into culture tubes. Wild plants may carry adult insects, larvae, pupae, or eggs on their surface or in their tissues. If these insects are used for culture without treatment, they may hatch or develop in the culture tube, interfering with the target fruit flies and affecting the observation and statistics of data such as egg production, hatching time, pupation time, and number of adults.

[0007] Furthermore, natural plants and fruits are prone to fermentation or mold growth in humid and relatively enclosed cultivation environments. Fermented food can contaminate the walls of the culture tubes, affecting the observation of eggs, larvae, pupae, and adults. Mycelia on the surface of fruits can also entangle or trap fruit flies, restricting their movement or even causing death. When there is too much water in the culture tube, large water droplets easily form on the tube walls. Adults' wings may get stuck when they come into contact with these droplets, affecting their activity, mating, or causing death. Conversely, when there is insufficient water in the culture tube, the plant substrate is prone to dehydration, which is not conducive to maintaining a suitable cultivation environment.

[0008] Therefore, it is necessary to provide a laboratory rearing method adapted to the specialized diet of the target fruit fly, which can simultaneously meet the oviposition and larval feeding needs of the target fruit fly by using natural host plants without the need to prepare complex artificial culture media, meet the nutritional needs of the adult fruit fly by using readily available fruits, and improve the culture stability of the target fruit fly by using culture containers, filter paper support layers, host plant pretreatment, culture environment control and daily maintenance measures, so as to achieve continuous generation maintenance of the target fruit fly under laboratory conditions. Summary of the Invention

[0009] The purpose of this invention is to solve the problem that specialized fruit flies that rely on specific natural host plants are difficult to complete egg laying, hatching, larval development, pupation and emergence in the laboratory using conventional methods, thus making it difficult to form a stable and continuous population.

[0010] This invention also aims to address the problem that host plants collected in the wild may carry the eggs of other insects, interfering with experimental culture, and to solve the problem that natural host plants and fruits, after fermentation, mold growth, contamination of the tube walls, or formation of mycelium in the culture tube, affect the survival of fruit flies and experimental observation.

[0011] The present invention further aims to solve the problem of difficult humidity control inside the culture tube, so that the host plant substrate can be kept in a state suitable for the target fruit fly to lay eggs and for the larvae to feed, while avoiding excessive water droplets adhering to the adult wings on the tube wall.

[0012] This invention also aims to provide a simple, readily available, and efficient rearing method that requires fewer steps and eliminates the need for additional artificial culture media. This method allows researchers to observe the egg-laying of target fruit flies under a stereomicroscope and record developmental indicators such as hatching, pupation, emergence, and sexual maturity, providing stable experimental materials for genetics, developmental biology, and other related experiments.

[0013] To address the aforementioned technical problems, this invention provides a method for rearing specialized fruit flies that rely on specific natural host plants. The method first involves collecting fruit flies and host plants based on the target fruit fly's natural habitat and host relationship. Then, the host plants are subjected to low-temperature treatment to prepare a plant substrate suitable for oviposition and larval feeding. This plant substrate, along with adult food, is then placed together in a culture tube lined with moistened filter paper.

[0014] The target fruit fly refers to a fruit fly whose larvae rely on a specific natural host plant for feeding and development, and whose female adults preferentially lay eggs on the natural host plant. The specific natural host plant serves as both the oviposition site for the female adults and the food for the hatched larvae.

[0015] Taking a method for rearing specialized fruit flies that rely on a specific natural host plant as an example, the target fruit fly is *Hymenochloa chinensis*, and the target fruit flies generally live in shrublands near residential areas along streams where *Hymenochloa chinensis* grows in abundance. Female adult flies can be obtained by scanning the *Hymenochloa chinensis* with a net. After identifying the collected samples, fertilized female adults are transferred to laboratory culture containers using a suction tube.

[0016] When establishing a single female line, a fertilized female adult collected from the wild is placed in each culture tube, and the offspring produced by the fertilized female adult form the corresponding single female line. Since the fertilization status of wild female adults may be different, there may be differences in the oviposition start time, visible oviposition amount and number of offspring between the single female lines.

[0017] After the wild-collected hyacinths were sorted and packaged, they were frozen in an ultra-low temperature freezer. The freezing treatment was used to kill the eggs of other insects that may be carried on or in the hyacinths, thereby reducing the impact of miscellaneous insects on the cultivation and observation of the target fruit flies.

[0018] The present invention can freeze hyacinth at ultra-low temperature for 1 to 2 weeks. As a preferred embodiment, hyacinth is placed in an ultra-low temperature freezer at -80°C for two weeks. After freezing, the hyacinth is taken out and allowed to return to room temperature before being used for cultivation, thus avoiding direct contact between the hyacinth in a low-temperature state and the target fruit flies.

[0019] To accommodate both adult egg-laying and larval feeding, the hyssop, now at room temperature, was divided into two parts. One part was cut into 0.5–1 cm segments to preserve some of the plant tissue structure. The other part was ground using a soymilk maker or similar equipment to create finer plant material. The hyssop segments and the ground hyssop were then mixed in a 1:1 ratio to form the hyssop host plant substrate.

[0020] The fragments of hyssop can form an oviposition and activity space with certain gaps and a three-dimensional structure. The crushed hyssop makes it easier for larvae to access and feed on plant tissues. When the two are mixed, the tissue characteristics of the natural host plant are preserved, and the uniformity of the distribution of plant material in the culture tube is improved.

[0021] Preferably, the culture tube has a diameter of 2 cm and a height of 9.5 cm. The culture tube should be cleaned and thoroughly dried before use to prevent residual dirt or excessive cleaning water from affecting the culture environment. The opening of the culture tube should be sealed with a cylindrical sponge plug to prevent fruit flies from escaping while maintaining gas exchange between the inside and outside of the tube. Transparent and easily observable culture tubes should be selected as the rearing container.

[0022] Two layers of filter paper are laid at the bottom of the culture tube and moistened with water. The two layers of filter paper form a filter paper support layer with a height of about 2cm to 3cm inside the culture tube. The filter paper serves two purposes: firstly, it supports the host plant substrate of hyssop, reducing the direct flow of plant fermentation liquid or fruit juice to the bottom of the culture tube; secondly, it maintains a humid environment inside the culture tube, preventing the host plant substrate from losing water too quickly.

[0023] Filter paper can also reduce the direct contamination of the culture tube wall by fermented food, keeping the culture tube wall relatively clean and making it easier for researchers to observe the activity and development of adults, larvae and pupae from the outside of the culture tube.

[0024] Use tweezers to pick up the prepared room temperature hyssop host plant substrate and place it on the moist filter paper inside the culture tube. The amount of hyssop host plant substrate should be sufficient to provide an oviposition site for female adults and meet the feeding needs of larvae.

[0025] A small piece of apple and a small piece of kiwi are placed in the culture tube. The apple and kiwi pieces mainly provide sugar, vitamins and other nutrients for the adults to maintain their activity, mating and egg laying. The host plant substrate of hyssop is placed together with the apple and kiwi pieces in the culture tube to meet the main nutritional needs of the larvae and adults respectively.

[0026] In the first generation establishment stage, fertilized female adults collected and identified in the field are placed into the above-mentioned culture tubes. When it is necessary to establish a single female line, one fertilized female adult is placed in each culture tube and the culture tubes are numbered; when it is necessary to maintain offspring, female and male offspring adults can be placed into culture tubes in a predetermined ratio required for the experiment for pairing and culture.

[0027] Culture tubes containing target fruit flies and food were placed in a temperature of 22°C, relative humidity of 75%, and a daily light exposure of 12 hours. The remaining time, excluding the light exposure period, was designated as the non-light exposure period to establish a stable diurnal cycle.

[0028] Observe daily during cultivation. Apple and kiwi pieces have high moisture content and are prone to mold growth. When mold spots or signs of mold appear on the apple or kiwi pieces, they should be replaced promptly to prevent mycelium from continuing to grow and trapping the target fruit flies.

[0029] If mold spots appear in other parts of the culture tube, use cotton soaked in anhydrous ethanol to wipe them off. When wiping, avoid allowing large amounts of anhydrous ethanol to come into contact with the target fruit flies and the hyssop host plant substrate that serves as food for the larvae.

[0030] Check the water droplets on the walls of the culture tubes daily. When the humidity inside the culture tubes is high or the temperature changes, water droplets may appear on the tube walls. A small amount of water is helpful in maintaining a humid environment, but when there are many water droplets or the water droplets are large enough to stick to the wings of adult insects, the excess water droplets should be wiped off in time to prevent the wings of adult insects from being stuck and unable to move normally, or even causing death.

[0031] During cultivation, a stereomicroscope can be used to observe oviposition on the host plant substrate of *Hyssopus chinensis*, and the start time and number of observable eggs can be recorded. Since some eggs may be laid inside the crushed *Hyssopus chinensis* substrate and cannot be directly observed from the substrate surface, the number of eggs recorded under the microscope can be considered as the visible oviposition amount, and the actual oviposition amount may be higher than the visible oviposition amount.

[0032] Continue to observe and record the hatching and emergence time of larvae, larval activity and feeding, pupation time, pupa emergence, emergence time, number of emergence, and the status of male and female adults. You can also record the time interval between emergence and mating of offspring adults to determine the sexual maturity time of offspring.

[0033] In a typical culture observation, the target fruit flies usually lay eggs 3 to 4 days after the start of culture, observe larvae on day 8 to 9, observe pupae on day 13 to 15, and observe adult emergence on day 18 to 20. The fertilization status of different females, the start time of egg laying, the culture batch, and the starting point of observation may affect the corresponding records.

[0034] After the adult offspring emerge, female and male adults are paired according to the experimental design. They are then placed in culture tubes containing moistened filter paper, hyssop host plant substrate, apple pieces, and kiwi pieces in a predetermined ratio. The culture is then continued under the above-mentioned culture conditions and maintenance methods to complete the reproduction of the next generation.

[0035] By continuously providing treated hyssop host plant substrate, apple chunks, and kiwi chunks, and managing moldy food and water in the culture tubes, the above process can be repeated generation by generation to achieve continuous maintenance of the target fruit fly population in the laboratory. Beneficial effects

[0036] Compared with culture methods that do not consider the host dependence of specialized fruit flies, the present invention has at least the following beneficial effects.

[0037] 1. Based on the characteristic that the target fruit fly larvae rely on natural host plants, this invention uses specific natural host plants such as hyssop as both the oviposition site for female adults and the food for larvae, so that the rearing environment is adapted to the natural diet of the target fruit fly, which is conducive to the target fruit fly completing the process of oviposition, hatching, larval feeding, pupation and emergence.

[0038] 2. This invention uses ultra-low temperature freezing to pretreat host plants collected in the field, which can kill or reduce the number of other insect eggs carried by the host plants, and reduce the interference of other insects on the target fruit flies and experimental observation after hatching in the culture tube.

[0039] 3. Cut a portion of the hyssop into 0.5-1cm segments, and crush the other portion of the hyssop and mix them together in a 1:1 ratio. This approach balances the plant tissue structure with the ease of feeding for larvae, making the hyssop substrate suitable for use as a place for oviposition and larval growth and development in the culture tube.

[0040] 4. Place two layers of moist filter paper at the bottom of the culture tube. This can support the host plant and fruit pieces, reduce the contamination of the culture tube wall by fermented food, and help maintain a moist environment inside the culture tube. At the same time, it makes it easier for researchers to observe the target fruit flies through the relatively clean culture tube wall.

[0041] 5. The host plant substrate of *Hylocereus undatus* mainly meets the feeding needs of larvae and the oviposition needs of female adults, while apple and kiwi pieces mainly provide adults with nutrients such as sugar and vitamins. Different food materials meet the needs of different developmental stages of the target fruit flies.

[0042] 6. This invention does not require the preparation of complex artificial culture media. Achyranthes bidentata is widely distributed, and apples and kiwis are also readily available. Therefore, it has the advantages of convenient material selection, simple culture equipment, and easy-to-master operation steps.

[0043] 7. By promptly replacing moldy fruit, wiping mold spots with cotton soaked in anhydrous ethanol, and removing excess water droplets from the culture tube walls, the likelihood of mycelia trapping the target fruit flies and water droplets sticking to the adult flies' wings can be reduced, thus improving the stability of the culture process. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the experimental feeding device of the present invention; Figure 2 This is a schematic diagram of the host plant *Achyranthes bidentata* used in this invention; In the picture: 1. Sponge plug; 2. Fruit fly; 3. Achyranthes bidentata; 4. Culture tube; 5. Apple and kiwi pieces; 6. Filter paper.

[0045] The culture tube is used to contain the target fruit flies and feeding materials; a sponge plug is placed at the opening of the culture tube; filter paper is placed at the bottom of the culture tube; hyssop is placed on the moistened filter paper; apple and kiwi pieces are placed together with hyssop in the culture tube; the target fruit fly adults complete feeding, mating and egg laying in the culture tube. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The following embodiments are all based on the disclosed technical content and are used to illustrate the implementation process of the present invention; they should not be construed as undue limitation on the scope of protection of the present invention.

[0047] Example 1: Field material collection and establishment of a single female line The collection area was selected in the streamside woodland near the residential area where hyssop was growing. Since the target fruit flies live and move around near the host plants in this type of environment, the collection was carried out by sweeping nets above and between the hyssop plants.

[0048] The fruit fly samples obtained from the sweep net were identified, and female adult samples were selected. Female adults collected in the wild are generally already fertilized. Each female adult was collected separately using a siphon and transferred. During the collection process, mechanical damage to the wings, legs, and abdomen of the adults was avoided.

[0049] To establish a single female line, each fertilized female adult was placed in an independent culture tube. Each culture tube was assigned a corresponding number so that the eggs, larvae, pupae, and adults obtained subsequently could all be traced back to the corresponding wild female adult.

[0050] Single-line female culture is beneficial for observing the oviposition time, visible oviposition amount, number of offspring emerging, and sexual maturity time of different wild female adults, and provides culture materials with a clear source for subsequent genetic and developmental studies.

[0051] Example 2: Preparation of host plant substrate for Achyranthes bidentata Fresh hyacinth chrysanthemums were collected from the wild, sorted and packaged, and then frozen in an ultra-low temperature freezer at -80°C for two weeks. This long-term ultra-low temperature treatment killed any other insect eggs carried by the hyacinth chrysanthemums.

[0052] After freezing, remove the hyssop from the ultra-low temperature freezer and place it at room temperature until it returns to room temperature. Once it has returned to room temperature, cut, crush, and cultivate it for use.

[0053] Divide the hyssop that has been brought back to room temperature into two parts of similar size or weight. Cut the first part of the hyssop into segments of 0.5 to 1 cm in length. The segments include tender leaves and stems that can be fed to the target fruit fly larvae.

[0054] The second part of the hyssop was put into a soymilk maker and crushed to form fine plant material from the tender leaves and stems of the hyssop. The hyssop pieces and the crushed hyssop were mixed evenly in a 1:1 ratio to obtain the hyssop host plant substrate.

[0055] In the mixed host plant substrate of *Hypericum hyssop*, segments of *Hypericum hyssop* with a length of 0.5–1 cm can provide a certain plant structure and gaps, while the crushed *Hypericum hyssop* material can increase the exposure of plant tissues, making it easier for newly hatched larvae to contact and feed.

[0056] Example 3: Setup of the culture device Select transparent culture tubes with a diameter of 2cm and a height of 9.5cm. Clean the culture tubes before use to remove any residue and allow them to dry thoroughly. Fit the tube opening with a cylindrical sponge plug that matches the tube size.

[0057] After cleaning and drying, place two layers of filter paper at the bottom of the culture tube and moisten the filter paper with water. The moistened filter paper forms a filter paper support layer with a height of about 2 to 3 cm inside the culture tube.

[0058] The filter paper should be kept moist, but not so moist that it creates a large amount of free water at the bottom of the culture tube that would submerge the hyssop substrate or trap adult insects. The filter paper should be moist enough to maintain the humidity inside the culture tube and support the hyssop substrate.

[0059] Using tweezers, the *Hypericum pertussis* host plant substrate prepared in Example 2 was picked up and placed on moist filter paper. The *Hypericum pertussis* substrate served as both an oviposition site for fertilized female adults and food for larvae after hatching in the culture tube.

[0060] Take a small piece of apple and a small piece of kiwi and place them in the culture tube. The apple and kiwi pieces should be placed next to the hyssop substrate, but the fruit pieces should not be completely covered on the hyssop substrate, so that the adult insects can feed on them and it is easy to check whether the fruit is moldy later.

[0061] The fertilized female adult insects are placed into the prepared culture tube, and then the opening of the culture tube is sealed with a cylindrical sponge plug. The sponge plug can prevent the adult insects from escaping, while allowing gas exchange between the inside and outside of the culture tube.

[0062] like Figure 1 As shown, the culture tube forms the rearing space, with moistened filter paper at the bottom, and the host plant substrate of *Hypericum perforatum*, apple pieces, and kiwi pieces placed above the filter paper. Adult fruit flies move inside the culture tube, and a sponge plug is located at the opening of the culture tube.

[0063] Example 4: Cultivation conditions and daily management The culture tubes containing the target fruit flies were placed in a culture environment with a temperature of 22℃, a relative humidity of 75%, and a daily light exposure of 12h. During the culture period, the culture tubes were kept upright or in a state that prevented the internal food from coming into extensive contact with the tube wall.

[0064] Observe the adult insect activity, food status, water droplets on the tube wall, and mold growth in the culture tube every day. Apple and kiwi pieces are prone to mold. When mold spots or obvious mold are observed on the surface of the fruit, use tweezers to remove the moldy fruit piece and replace it with a new corresponding fruit piece.

[0065] When changing the fruit pieces, avoid injuring the target fruit flies and avoid damaging any eggs or larvae that may be present in the hyssop substrate. After the operation is completed, reseal the culture tube with a sponge plug.

[0066] When mold spots appear on the inner wall of the culture tube, around the filter paper, or in other areas where larvae are not feeding, use cotton soaked in anhydrous ethanol to wipe the mold spots. Control the amount of anhydrous ethanol used during wiping to prevent it from flowing into the hyssop substrate.

[0067] Observe the water droplets on the inner wall of the culture tube. If there are too many water droplets on the inner wall of the culture tube, and these droplets may come into contact with or adhere to the adult insect's wings, wipe them off promptly. By controlling the amount of water droplets, the risk of adult insects being unable to move normally or dying due to their wings getting stuck is reduced.

[0068] When the filter paper shows a clear tendency to dry out, it can be kept moist again without forming a large amount of water in the culture tube. By keeping the filter paper moist and removing water droplets from the tube wall in a timely manner, the culture tube can maintain a high relative humidity while avoiding excessive moisture.

[0069] Example 5: Observation of oviposition and development process The culture tubes were placed within the observation range of a stereomicroscope to observe the oviposition of fertilized female adults. The oviposition time after the start of culture and the number of eggs observable on the surface of the hyssop host plant substrate were recorded.

[0070] The eggs of the target fruit fly may be located on the surface of the hyssop segment or may be laid inside the crushed hyssop substrate. For eggs laid inside the substrate and not directly visible from the outside, the hyssop substrate should not be forcibly disturbed or damaged to avoid affecting egg hatching. Therefore, the recorded egg production mainly refers to the number of observable eggs.

[0071] In a series of culture observations, the target fruit flies generally began to lay eggs 3 to 4 days after the start of culture, observed larvae on days 8 to 9, observed pupae on days 13 to 15, and observed adult emergence on days 18 to 20.

[0072] Record the time of oviposition, larval emergence, pupa emergence and adult emergence for each culture tube. Also record the visible oviposition amount, final number of adults, number of male and female adults, and the time from adult emergence to mating for each culture tube.

[0073] During the observation process, the host plant substrate of *Hylocereus undatus* should not be frequently disturbed to avoid damaging the eggs and larvae in the substrate. When it is necessary to observe the larval activity, it can be observed through the wall of the transparent culture tube and the gaps between the *Hylocereus undatus* segments.

[0074] After observing the pupae, continue to culture them under the same conditions until the adults emerge. The adults continue to feed on apple and kiwi pieces in the culture tube until they reach sexual maturity and then pair up.

[0075] Example 6: Continuous maintenance of offspring After the first generation of adult insects emerged, healthy female and male adults were selected according to the required male-female ratio for the experiment. The host plant substrate of *Hypericum hyacinthum* was prepared again, and new culture tubes were set up according to the method in Example 3.

[0076] Selected female and male adults were placed in new culture tubes and allowed to mate and lay eggs under conditions of 22°C, 75% relative humidity, and 12 hours of light per day.

[0077] Inspect and replace the moldy apple and kiwi pieces according to the method in Example 4, scrub the mold spots in the culture tube, and wipe away any excess water droplets that may have adhered to the adult insect wings.

[0078] The egg-laying, larval hatching, pupation, and emergence of the second generation were observed and recorded according to the method in Example 5. After the second generation adults emerged and reached sexual maturity, male and female adults were selected again and placed in newly set culture tubes for the next generation of culture.

[0079] By repeating the steps of pretreatment with hyssop, preparation of host plant substrate, setting up culture tubes, introduction of adult flies, culture and maintenance, development observation and offspring pairing, the target fruit flies can be continuously bred and maintained in the laboratory.

[0080] Since this method does not rely on complex artificial culture media, and the main materials are hyssop, apple, kiwi, filter paper, culture tubes and sponge plugs, it is easy to set up multiple culture tubes at the same time according to the experimental material requirements, so as to realize the culture of multiple single female lines or multiple replicate groups.

[0081] Example 7: Record of Single Female Culture Four single female lines were cultured using the above method and numbered IEL1, IFL2, IFL3, and IFL4, respectively. The oviposition time, visible oviposition amount, larval emergence time, pupation time, number of adults, emergence time, and sexual maturity time of offspring were recorded for each single female line. The results are shown in Table 1.

[0082]

[0083] Table 1 The sexual maturity time in Table 1 is the time interval from the emergence of adult offspring to their ability to mate. The sexual maturity time of the offspring in the four single female lines is 5±2 days, indicating that the adult offspring obtained by this method can reach a state where they can continue to reproduce in a relatively short period of time.

[0084] The oviposition time of different monogynous lines is 1-2 days, with 10-20 eggs visible. The larvae appear in 6-7 days, pupation time is 12.5-13 days, emergence time is 16.5-17 days, and the number of emerging individuals is 30-45.

[0085] The specific records in Table 1 differ somewhat from the time ranges observed during general culture: oviposition at 3–4 days, larvae emerging at 8–9 days, pupae emerging at 13–15 days, and adulthood at 18–20 days. These differences can be recorded separately as actual culture results under different batches and observation timing conditions, and do not affect the fact that this result can be achieved at each stage within this culture system.

[0086] The fertilization status and the start time of oviposition may vary among wild-collected female adults; furthermore, some eggs may be located inside the substrate of *Hyssopus chinensis*, and the number of eggs and hatching time may not be fully detected in the first observation. Therefore, when using this method to conduct quantitative experiments, the incubation start time, observation interval, and recording standards should be standardized.

[0087] As shown in Table 1, all four single female lines produced adult offspring, with 30 to 45 adults emerging per tube. This indicates that the method can support the target fruit fly to complete the developmental process from oviposition, larval growth, pupation to adult emergence.

[0088] Once the adult offspring reach sexual maturity, they can be paired according to a predetermined male-to-female ratio and cultured using the same hyssop host plant substrate, apple chunks, kiwi chunks, moistened filter paper, and culture conditions to form a continuous laboratory population.

[0089] Example 8: Culture method for amplification of experimental materials When the experiment requires a large number of target fruit flies, multiple culture tubes can be set up simultaneously. Each culture tube is 2 cm in diameter and 9.5 cm in height, with two layers of moist filter paper at the bottom, and contains hyssop host plant substrate, apple pieces, and kiwi fruit pieces.

[0090] Depending on the experimental objective, single fertilized female adults can be used to establish breeding lines, or sexually mature male and female adults of the offspring can be placed together in a predetermined ratio in a culture tube. Different culture tubes are numbered separately, and information on oviposition, hatching, pupation, and emergence is recorded separately for each.

[0091] During multi-tube culture, check each tube for fruit mold and water droplets on the tube walls. If mold is found in an apple or kiwi piece in a culture tube, replace only the corresponding fruit piece in that culture tube to avoid mixing of hyssop substrate and fruit flies between different culture tubes.

[0092] For culture tubes that are needed as materials for genetic and developmental experiments, observation and selection should be carried out at the target developmental stage. For example, materials at the egg stage, larval stage, pupal stage, or adult stage can be obtained according to experimental needs; or single female lines that meet the experimental requirements can be selected based on culture records, such as oviposition time, number of emergence, or sexual maturity time.

[0093] When using a multi-tube parallel approach, the culture apparatus and operating methods remain consistent, which helps to reduce experimental deviations caused by differences in culture containers, food composition, and environmental conditions.

Claims

1. A method for raising specialized fruit flies that rely on specific natural host plants, characterized in that, Includes the following steps: S1. Obtaining fertilized female adult fruit flies, wherein the target fruit fly is a specialized feeding fruit fly whose larvae feed on a specific natural host plant and complete their growth and development on the specific natural host plant; S2. Collect the specific natural host plant, subject the specific natural host plant to low-temperature freezing treatment to kill the eggs of other insects carried by the specific natural host plant, restore the specific natural host plant to room temperature after the freezing treatment is completed, and crush at least a portion of the specific natural host plant to obtain a host plant substrate for the target fruit fly to lay eggs and for the larvae to feed. S3. Clean and dry the culture tube, place a water-moistened filter paper at the bottom of the culture tube, place the host plant substrate on the filter paper, and add apple pieces and kiwi pieces to the culture tube respectively. The host plant substrate is used as an oviposition site and larval food, and the apple pieces and kiwi pieces are used as adult food. S4. Seal the opening of the culture tube with the fertilized female adult insect and keep the inside and outside of the culture tube ventilated, and culture it under the set temperature, relative humidity and light cycle conditions; S5. During the cultivation process, the apple pieces and kiwi pieces are inspected and replaced, mold spots appearing in the cultivation tube are removed, and water droplets are wiped off when there are too many water droplets on the wall of the cultivation tube. S6. Observe and record the egg-laying, larval hatching, pupation and emergence of the target fruit fly, and obtain the offspring adults; S7. The offspring adults are placed in the culture tubes treated in steps S2 and S3 according to a predetermined male-female ratio and cultured for a longer period, thereby achieving continuous generation maintenance of the target fruit fly under laboratory conditions.

2. The method for raising specialized fruit flies that rely on specific natural host plants according to claim 1, characterized in that: In step S1, sweeping nets are used to collect fruit flies from the streamside woodland near the residential area where the specific natural host plants are dense. The collected fruit flies are identified, and fertilized female adults are collected using a siphon. A single female line is established using one fertilized female adult as the establishing individual.

3. The method for raising specialized fruit flies that rely on specific natural host plants according to claim 1, characterized in that: The target fruit fly is a fruit fly that feeds on the tender leaves and stems of *Hylocereus undatus* and uses *Hylocereus undatus* as its oviposition site. The specific natural host plant is *Hylocereus undatus*. The low-temperature freezing treatment in step S2 involves freezing the collected *Hylocereus undatus* at -80°C for 1 to 2 weeks.

4. The method for raising specialized fruit flies that depend on specific natural host plants according to claim 3, characterized in that: After being frozen and then brought back to room temperature, the hyssop was divided into two parts. One part was cut into hyssop segments of 0.5cm to 1cm in length, and the other part was crushed. The hyssop segments and the crushed hyssop were mixed in a 1:1 ratio to form the host plant substrate.

5. The method for raising specialized fruit flies that rely on specific natural host plants according to claim 1, characterized in that: The culture tube has a diameter of 2cm and a height of 9.5cm, and the opening of the culture tube is sealed with a cylindrical sponge plug; two layers of filter paper are placed inside the culture tube, and the moistened filter paper forms a filter paper support layer with a height of 2cm to 3cm inside the culture tube.

6. The method for raising specialized fruit flies that depend on specific natural host plants according to claim 1, characterized in that: The culture conditions in step S4 are a temperature of 22°C, a relative humidity of 75%, and a daily light exposure of 12 hours.

7. The method for raising specialized fruit flies that depend on specific natural host plants according to claim 1, characterized in that: In step S5, when the apple or kiwi piece shows signs of mold, the corresponding fruit piece is replaced; when mold spots appear in the culture tube, the mold spots are wiped with cotton soaked in anhydrous ethanol; when the water droplets on the culture tube wall reach a level that can adhere to the adult insect's wings, the water droplets are wiped off.

8. The method for raising specialized fruit flies that depend on specific natural host plants according to claim 1, characterized in that: In step S6, the oviposition situation is observed under a stereomicroscope, and the oviposition time and number of eggs, the time of larval emergence, the time of pupa emergence, the time of eclosion, the number of eclosions, and the sexual maturity time of the offspring adults are recorded respectively.

9. The method for raising specialized fruit flies that depend on specific natural host plants according to claim 1, characterized in that: The target fruit flies lay eggs 3-4 days after the start of the culture, larvae appear 8-9 days after the start of the culture, pupae appear 13-15 days after the start of the culture, and adults appear 18-20 days after the start of the culture. After the adult offspring emerge, they pair up and continue the culture for the next generation according to steps S2 to S7.

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