A high-throughput method for measuring fruit fly feed intake and its application

Abstract

This application provides a high-throughput method for measuring fruit fly feed intake and its application, belonging to the field of animal feed intake research. The method includes: transferring each of multiple fruit flies to be tested into each well of a feeding plate in a high-throughput fruit fly feeding platform; adding a liquid food with a predetermined chemical composition to each well of the food substrate of the high-throughput fruit fly feeding platform; allowing the multiple fruit flies to feed freely for a predetermined time; measuring the protein content in the remaining liquid food after free feeding to obtain the remaining protein content in each well; and obtaining the feed intake of each fruit fly based on the remaining protein content in each well. By combining a high-throughput fruit fly feeding platform with protein concentration measurement, changes in the protein content of the liquid food are tracked to estimate changes in liquid volume, thereby improving the efficiency and accuracy of fruit fly feed intake measurement while simultaneously achieving high-throughput measurement.

Description

Technical Field

[0001] This application relates to the field of animal feed intake research technology, and in particular to a high-throughput method for measuring feed intake in fruit flies and its application. Background Technology

[0002] The fruit fly (Drosophila melanogaster), as a classic model organism, has been widely used in fields such as nutrient metabolism, drug screening, and aging research. Studies of fruit fly feeding behavior are of great significance in revealing the relationship between nutrient intake and life processes.

[0003] Currently, various methods have been used to study the feeding behavior of fruit flies, reflecting their feeding characteristics from different levels and perspectives. The dye method uses dyes in food to label it, and the amount of dye is measured to infer the amount of food consumed by the fruit flies. The radioactive isotope labeling method uses radioactive isotopes to label food, and the isotope content is measured to reflect the amount of food consumed. The capillary glass tube feeding method... The first method (FLIC) indirectly measures fruit fly feeding by measuring the volume change of liquid food in a capillary glass tube. In addition, methods such as FLIC and flyPAD have been developed in recent years, which automate the measurement of fruit fly feeding by converting electrical signals. Although these methods can provide quantitative data to some extent, the accuracy is often affected by factors such as food evaporation and sample loss due to the complexity of fruit fly feeding behavior, and these methods show significant limitations when processing large numbers of samples. Therefore, developing a high-throughput, efficient, and accurate method for measuring fruit fly feeding has become an urgent need in current research. Summary of the Invention

[0004] This application provides a high-throughput method for measuring fruit fly feed intake and its application, in order to solve the following technical problem: how to improve the efficiency and accuracy of fruit fly feed intake measurement while simultaneously measuring it in high throughput.

[0005] In a first aspect, this application provides a method for determining the amount of food consumed by fruit flies, the method comprising:

[0006] Each of the multiple fruit flies to be tested is transferred to each hole of the feeding plate of the high-throughput fruit fly feeding platform;

[0007] Liquid food with a set chemical composition is added into each hole of the food base plate of the high-throughput fruit fly feeding platform;

[0008] The test fruit flies were allowed to feed freely for a set period of time.

[0009] The protein content of the remaining liquid food in each well of the food substrate after free feeding was determined to obtain the remaining protein content in each well; and

[0010] The amount of food consumed by each fruit fly was determined based on the amount of remaining protein in each well.

[0011] Optionally, the specified chemical components include: food-grade surfactants.

[0012] Optionally, the mass of the edible surfactant is 0.1% to 1% of the total mass of the liquid food, and the edible surfactant includes one or more of Tween 20, Tween 80, and polyethylene glycol.

[0013] Optionally, the specified chemical components may also include: sucrose, yeast extract, propionic acid, sodium benzoate, and solvent.

[0014] Optionally, determining the protein content of the remaining liquid food in each hole of the food substrate includes:

[0015] Solvent from the remaining liquid food in each hole of the food base plate was removed to obtain multiple powder samples;

[0016] The protein content in the multiple powder samples was determined separately.

[0017] Optionally, the solvent in the remaining liquid food is removed from each hole of the food base plate by freeze drying, wherein the vacuum degree of freeze drying is <10 Pa and the freeze drying time is 18h to 24h.

[0018] Optionally, the determination of protein content in the plurality of powder samples is performed using the BCA method or the Bradford method.

[0019] Optionally, the amount of food consumed satisfies the following relationship:

[0020] I = (C0 - C1) / C0 × V

[0021] In the formula, I represents the amount of food consumed, C0 represents the protein content of the liquid food before consumption, C1 represents the protein content of the liquid food after consumption, and V represents the volume of the liquid food before consumption.

[0022] Optionally, the volume of liquid food in each hole of the food base plate is 10 μL to 30 μL.

[0023] Secondly, this application provides an application of the method described in any embodiment of the first aspect, the method being used to investigate the effects of adding different nutrients or drugs on the feeding behavior of fruit flies.

[0024] The technical solutions provided in this application have the following advantages compared with the prior art:

[0025] This application provides a method for determining the feed intake of fruit flies. The method includes: transferring each of a plurality of fruit flies to be tested into each well of a feeding plate of a high-throughput fruit fly feeding platform; adding liquid food with a set chemical composition to each well of the food substrate of the high-throughput fruit fly feeding platform; allowing the plurality of fruit flies to feed freely for a set time; measuring the protein content in the remaining liquid food in each well of the food substrate after free feeding to obtain the remaining protein content in each well; and obtaining the feed intake of each fruit fly based on the remaining protein content in each well. This application combines a high-throughput fruit fly feeding platform, a multi-well food substrate, and protein concentration measurement, and calculates the change in liquid volume by tracking the change in protein content in the liquid food, thereby improving the efficiency and accuracy of fruit fly feed intake measurement while performing high-throughput measurement. Attached Figure Description

[0026] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0027] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 A flowchart illustrating a method for determining the feed intake of fruit flies, provided as an embodiment of this application;

[0029] Figure 2 This is the high-throughput fruit fly feeding platform provided in Embodiment 1 of this application;

[0030] Figure 3 This is a schematic diagram of the method for determining the feed intake of fruit flies provided in Embodiment 1 of this application;

[0031] Figure 4 This is a diagram illustrating the actual application effect of the fruit fly feeding amount provided in Embodiment 1 of this application;

[0032] Figure 5 The survival rate of Oregon-R fruit flies under different dietary conditions is shown in Example 2 of this application.

[0033] Figure 6This is a graph showing the difference in daily food intake of Oregon-R fruit flies under different dietary conditions, as provided in Example 2 of this application.

[0034] Figure 7 This is a graph showing the effect of 36 types of flavonoid compounds provided in Example 3 of this application on the daily food intake of fruit flies. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0037] Furthermore, in the description of this application, the terms "comprising," "including," etc., mean "including but not limited to." In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In this document, "and / or" describes the relationship between related objects, indicating that three relationships can exist; for example, A and / or B can represent: A alone, A and B simultaneously, or B alone. A and B can be singular or plural. In this document, "at least one" means one or more, and "more than" means two or more. "At least one," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c" or "at least one of a, b, and c" can both represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple. "Parts representation," such as parts by weight or parts by mass, indicates the proportional relationship between components. In the proportional relationships discussed in this article, parameters described by proportion should be understood as the first term of the proportion in the order of description, while the proportion figure should be understood as the second term. For example, if the mass ratio of substance A, substance B, and substance C is 1:2:3, then substances A, B, and C should correspond one-to-one with the proportion figure in the proportion in the order of description, i.e., the mass of substance A : the mass of substance B : the mass of substance C = 1:2:3.

[0038] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0039] Figure 1 This is a flowchart illustrating a method for determining the food intake of fruit flies, as provided in an embodiment of this application.

[0040] This application provides a method for determining the amount of food consumed by fruit flies, such as... Figure 1 As shown, the method includes:

[0041] S1. Transfer each of the multiple test fruit flies to each hole of the feeding plate of the high-throughput fruit fly feeding platform.

[0042] The high-throughput fruit fly feeding platform, through its integrated design, enables precise control of the fruit fly rearing environment, including temperature, humidity, and light conditions—all key factors affecting fruit fly growth and development. More importantly, the platform supports large-scale, multivariate experimental designs, enabling simultaneous experiments with multiple variables (such as the addition of different nutrients or drugs), making it suitable for large-scale screening experiments.

[0043] Feeding boards are components in high-throughput fruit fly feeding platforms used to hold fruit flies. They typically have multiple small holes or channels, each capable of holding one or more fruit flies.

[0044] S2. Add liquid food with a set chemical composition into each hole of the food base plate of the high-throughput fruit fly feeding platform;

[0045] It should be noted that, in the embodiments of this application, the food substrate can be a standard 96-well plate, used to place and provide the liquid food required by the fruit flies. In the high-throughput fruit fly feeding platform, the position of the holes on the feeding plate matches that of the 96-well plate, ensuring that the food can be evenly distributed to the location of each fruit fly.

[0046] Steps S1 and S2 ensure that each fruit fly has its own feeding space, avoiding mutual interference between fruit flies.

[0047] In some embodiments, the specified chemical components include: food-grade surfactants.

[0048] Optimizing the liquid food formulation is crucial for ensuring normal feeding behavior in fruit flies. The chemical composition of the liquid food can be customized according to experimental needs to study the effects of different components on fruit fly feed intake. This application introduces an edible surfactant into the liquid food, effectively reducing its viscosity. The innovations of this measure include: ① Improved fruit fly feeding behavior: Lower viscosity makes feeding smoother for fruit flies, avoiding feeding difficulties caused by excessive viscosity, thereby improving the naturalness of feeding behavior and the accuracy of data. ② Experimental repeatability and precision: The addition of an edible surfactant prevents excessive adhesion of the liquid food to the feeding plate, ensuring consistency in food volume and feed intake measurements in each experiment, thus improving experimental repeatability and precision.

[0049] In some embodiments, the mass of the edible surfactant is 0.1% to 1% of the total mass of the liquid food, and the edible surfactant includes one or more of Tween 20, Tween 80, and polyethylene glycol.

[0050] Tween 20, Tween 80, and polyethylene glycol can reduce the viscosity and surface tension of liquid foods. Furthermore, limiting the mass of the edible surfactant to 0.1% to 1% of the total mass of the liquid food has no significant impact on the health of fruit flies and ensures food safety. For example, the mass of the edible surfactant can be 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, or 1% of the total mass of the liquid food.

[0051] In some embodiments, the specified chemical components further include: sucrose, yeast extract, propionic acid, sodium benzoate, and solvent.

[0052] Sucrose is the main carbon source in the liquid diet of fruit flies, providing them with energy.

[0053] Yeast extract is an important nitrogen source in the liquid diet of fruit flies, providing them with essential amino acids and other nutrients.

[0054] Propionic acid primarily acts as a pH buffer in the liquid diet of fruit flies. During feeding, fruit flies produce carbon dioxide, which acidifies the food. Propionic acid helps regulate the pH of the food, maintaining it within a suitable range for the fruit flies.

[0055] Sodium benzoate is primarily used as a preservative in fruit fly liquid food. It inhibits bacterial growth and extends the shelf life of the food.

[0056] S3. Allow the multiple fruit flies to be tested to feed freely within a set time period;

[0057] S4. Measure the protein content of the remaining liquid food in each hole of the food substrate after free feeding, and obtain the remaining protein content of each hole;

[0058] In some embodiments, determining the protein content of the remaining liquid food in each hole of the food substrate includes:

[0059] Solvent from the remaining liquid food in each hole of the food base plate was removed to obtain multiple powder samples;

[0060] The protein content in the multiple powder samples was determined separately.

[0061] This application calculates the change in liquid volume by tracking changes in the protein content of liquid food, thereby accurately measuring the amount of food consumed by fruit flies. It solves the problems of feeding behavior interference, liquid evaporation loss, and difficulty in achieving high throughput in existing measurement methods such as dye method and capillary feeding method.

[0062] In some embodiments, the solvent in the remaining liquid food is removed from each hole of the food base plate by freeze drying, wherein the freeze drying vacuum degree is <10 Pa and the freeze drying time is 18 h to 24 h.

[0063] Freeze-drying technology is used to remove residual moisture from the food substrate after fruit flies have ingested the food, avoiding differences in liquid volume per pore caused by variations in feeding behavior among individual fruit flies. Compared with traditional air-drying methods, it has the advantages of easy dissolution and rehydration, and extended shelf life. For example, the freeze-drying time can be 18h, 19h, 20h, 21h, 22h, 23h, 24h, etc.

[0064] In some embodiments, the determination of protein content in the plurality of powder samples is performed using the BCA method or the Bradford method.

[0065] It should be noted that the BCA (Bicinchoninic acid) method is a protein concentration determination method based on the biuret reaction. Under alkaline conditions, proteins will react with Cu... 2+ Reduced to Cu + It forms a purple-blue complex with BCA reagent. This complex has a high absorbance at 562 nm, and the amount of the reaction product is directly proportional to the protein concentration. The Bradford method is a protein concentration determination method that utilizes the significant color change that occurs when protein binds to Coomassie brilliant blue G250 (or Coomassie brilliant blue G-250). Under acidic conditions, protein binds to Coomassie brilliant blue G250 to form a stable blue complex. The maximum absorption peak of this complex shifts from 465 nm to 595 nm, and the extinction coefficient increases. By measuring the absorbance at 595 nm, the protein concentration can be calculated.

[0066] Estimating fruit fly feed intake by measuring residual protein content in liquid food has the following advantages: ① Avoids dye interference: The BCA and Bradford methods do not require the addition of extra dyes to the food, avoiding potential interference from dyes on fruit fly feeding behavior. ② High sensitivity and accuracy: The BCA and Bradford methods can detect extremely low concentrations of protein, with significantly higher accuracy and sensitivity than dye methods, ensuring the accuracy of feed intake determination. ③ Data consistency: This method can accurately measure differences in feed intake between different samples without affecting the normal feeding behavior of fruit flies, providing high-quality data support for subsequent physiological and metabolic studies.

[0067] S5 determines the amount of food consumed by each fruit fly based on the remaining protein content in each well.

[0068] In some implementations, the amount of food consumed satisfies the following relationship:

[0069] I = (C0 - C1) / C0 × V

[0070] In the formula, I represents the amount of food consumed, C0 represents the protein content of the liquid food before consumption, C1 represents the protein content of the liquid food after consumption, and V represents the volume of the liquid food before consumption.

[0071] In some embodiments, the volume of liquid food in each hole of the food base plate is 10 μL to 30 μL.

[0072] Methods for analyzing fruit fly feeding results: ① Precise measurement of feeding amount: Precise measurement of feeding amount can be performed using statistical software (Graphpad Prism 9) to compare differences in feeding amount under different experimental conditions. ② Application of experimental results: The experimental results can be used to study the effects of different nutrients, drugs, or other treatments on fruit fly feeding behavior. ③ Data reliability and repeatability: To improve the reliability and repeatability of the results, the experiment needs to be repeated multiple times, and statistical analysis should be performed based on the differences in feeding amount between different treatment groups.

[0073] In summary, this application achieves precise measurement of liquid food intake in fruit flies by using a high-throughput fruit fly feeding platform combined with protein concentration determination. Compared with existing methods such as dye methods and capillary feeding methods, this invention demonstrates significant advantages in efficiency and accuracy, specifically with the following beneficial effects:

[0074] 1. High throughput and high efficiency: This invention utilizes a high-throughput fruit fly feeding platform, which can process multiple fruit flies simultaneously, significantly improving experimental throughput and efficiency. This large-scale, simultaneous processing capability is particularly suitable for experiments requiring large amounts of data, such as nutritional research and drug screening. Compared to traditional single-fly feeding methods, this platform can obtain a large amount of data in a short time, effectively reducing experimental cycles and labor intensity.

[0075] 2. High-precision food intake measurement: By using a protein concentration determination method, this invention avoids the interference that traditional dye methods may cause to fruit fly feeding behavior. Dye methods require the addition of extra chemicals, which may affect the fruit flies' feeding preferences, leading to data distortion. The protein content in liquid food is not affected by evaporation during the experiment. Compared to dye or capillary methods, this invention uses a standard curve to accurately calculate the fruit fly's food intake, eliminating the interference of external factors on the measurement and ensuring the stability and accuracy of the data.

[0076] 3. Optimization of liquid food formulation: This invention adds surfactants to the liquid food, which effectively reduces the viscosity of the liquid food and prevents excessively high food viscosity from adversely affecting the fruit flies' feeding and survival, such as difficulty in feeding or accidental ingestion of food residue on the feeding board; it also prevents high-viscosity liquid from excessively adhering to the feeding board, affecting the accuracy of food intake measurement, and further improves the reliability of experimental data.

[0077] 4. Data Support and Application Expansion: This invention can not only be applied to basic nutrition research, but also provide efficient and accurate data support for drug screening and metabolic studies. By accurately measuring the feeding behavior and trends of fruit flies under different diets or drug treatments, the effects of different components on fruit fly metabolism, lifespan, and health can be effectively analyzed. This precise data support helps to better understand the mechanisms of action of nutrients and drugs, and provides a reference model for human health research.

[0078] Based on a general inventive concept, this application provides an application of the method described in any of the above embodiments, the method being used to investigate the effects of adding different nutrients or drugs on the feeding behavior of fruit flies.

[0079] By utilizing a high-throughput fruit fly feeding platform (WAFFL) and protein concentration detection method, this application enables the simultaneous determination of fruit fly feed intake under multiple variable conditions, such as the addition of different nutrients or drugs, thereby meeting the needs of efficient large-scale experiments.

[0080] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to industry standards. If there is no corresponding industry standard, then common international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0081] Experimental equipment and materials:

[0082] 1. WAFFL: A high-efficiency fruit fly feeding platform designed using 96-well cell culture plates, which can accurately monitor the feeding activities of multiple fruit flies and is suitable for research fields such as nutrition and drug screening.

[0083] 2. 96-well cell culture plate: Standardized 96-well plates are used to process multiple fruit fly samples at the same time, with each well for feeding only a single fruit fly.

[0084] 3. Freeze dryer: Used to remove moisture from the liquid food consumed by fruit flies, avoiding interference from differences in feeding among individual fruit flies on the results of feeding amount measurement, and improving the accuracy and reliability of the experiment.

[0085] 4. Fruit fly liquid food: including 2.5% sucrose, 5% yeast extract, 0.3% propionic acid, 0.1% sodium benzoate, 0.5% Tween 20, and the remainder is water.

[0086] 5. BCA Protein Concentration Assay Kit: Used to determine the protein concentration in liquid food before and after ingestion.

[0087] 6. Microplate reader: Quantifies the protein concentration in each well of a 96-well plate at an absorbance of 562 nm.

[0088] Example 1

[0089] This embodiment provides a method for determining the food intake of Drosophila. This method combines WAFFL (High-throughput Drosophila Feeding Platform), 96-well cell culture plates, and BCA protein concentration measurement. The method specifically includes the following steps:

[0090] Step 1: Preparation of Liquid Food: Mix all ingredients in the liquid food formula thoroughly, ensuring the solution contains no visible particles or insoluble substances. Filter the food using a filter to ensure its cleanliness and stability. The composition of the liquid food can be flexibly adjusted according to experimental needs, for example, by adding specific drugs or nutrients to test its effects on fruit fly feeding behavior.

[0091] Step 2, Preparation of Fruit Flies: Use healthy and homogeneous fruit fly strains for experiments, typically wild-type fruit flies (Oregon R) or other commonly used laboratory fruit fly strains. Group the fruit flies according to sex, age, and weight to ensure data consistency and experimental comparability.

[0092] Step 3: Feeding the fruit flies: The fruit flies are fed using a WAFFL (Whole Air Flying Board). Each well of the 96-well food tray is for the exclusive use of the corresponding single fruit fly in the feeding tray to prevent interference between flies. Add 20 μl of liquid food to each well, ensuring the liquid is evenly distributed at the bottom of the well. Depending on the specific experimental design, the fruit flies can freely feed in the chamber for a certain period (e.g., 24 hours).

[0093] Step 4: Remove residual moisture from the well plate after fruit fly feeding: After the prescribed feeding time, slowly remove the 96-well plate and place it in a -80°C freezer for pre-cooling (1-2 hours). After the vacuum degree of the freeze dryer reaches below 10 Pa, continue to run it for 18-24 hours until the sample in the well plate is powdery.

[0094] Step 5, BCA protein concentration determination: Add 100 μl of PBS (diluted 5 times) to each well of the lyophilized 96-well plate. Using the BCA protein concentration detection kit, prepare the standard solution and BCA working solution. Take 10 μl of the diluted sample from each well into another 96-well plate, add 100 μl of BCA working solution to each well, mix well, incubate at 37°C for 30 minutes, and measure the absorbance at 562 nm using a microplate reader. Measure the absorbance of each well.

[0095] Step 6: Calculation of fruit fly feed intake: Plot a standard curve based on protein standards of known concentrations. By comparing the absorbance changes of each well before and after fruit fly feeding with the standard curve, the protein concentration of each well can be obtained. Based on the ratio of protein amount to liquid volume, the feed intake of each fruit fly within a specific time period can be calculated.

[0096] ① Protein content per well (μg) = Protein concentration (μg / μl) × 10 μl × 10

[0097] ② Fruit fly food intake (μl) = [Pre-feeding protein content (μg) - Remaining protein content (μg)] / Pre-feeding protein content (μg) × Original liquid food volume (μl).

[0098] Figure 2 The components of the high-throughput fruit fly feeding platform provided in Embodiment 1 of this application. Figure 2 (a) WAFFL feeding plate. (b) Food is located in a 96-well plate beneath the WAFFL, allowing up to 96 parallel experiments in a single WAFFL. The top is sealed with a reusable silicone pad to maintain humidity. (c) An acrylic cap can be used instead of the silicone pad to visualize the behavior of the fruit flies during the experiment. (d) A transfer adapter and receiving plate are used to transfer the fruit flies while maintaining their position on the 96-well plate.

[0099] Figure 3 This is a schematic diagram of the method for determining fruit fly food intake provided in Embodiment 1 of this application. Figure 3 It can be seen that the daily food intake of fruit flies can be measured using the WAFFL platform, 96-well cell culture plates, and BCA protein concentration assay.

[0100] Figure 4 This is a diagram illustrating the actual application effect of the fruit fly feeding amount provided in Embodiment 1 of this application; Figure 4 In this study, (a) the evaporation of liquid food does not affect the protein content of the food; and (b) the volume of liquid food measured by this method is directly proportional to the protein content, with a correlation coefficient R. 2 (c) Accurately determine the compensatory feeding of fruit flies to different concentrations of food; (d) Accurately determine the amount of food consumed by fruit flies of different sexes and mating states.

[0101] Example 2

[0102] This embodiment studies the effects of different dietary components on fruit fly feeding behavior, metabolism, and health by accurately measuring the amount of food consumed by fruit flies under different dietary conditions, providing data support for understanding the mechanism of nutrition's effect on organisms.

[0103] Experimental Design:

[0104] 1. Experimental subjects: Male Oregon R fruit flies on the first day of emergence were selected and divided into three groups, with 96 fruit flies in each group.

[0105] 2. Dietary conditions: Control group (liquid food composition: 2.5% sucrose, 5% yeast extract, 0.3% propionic acid, 0.1% sodium benzoate, 0.5% Tween 20, with the remainder being water); high-sugar diet (sucrose content in food increased to 5%); high-protein diet (yeast extract content in food increased to 10%); high-fat diet (coconut oil added to food up to 20%).

[0106] 3. Experimental Procedure: In the WAFFL system, 20 μl of liquid food (corresponding to different dietary formulas) was added to each well of a 96-well plate. Only one fruit fly was placed in each well to ensure independent feeding. The fruit flies fed freely in their respective chambers, and the amount of food consumed was monitored regularly. From day 1 to day 7 of the experiment, the changes in the amount of food consumed were recorded daily. After each feeding, the 96-well plate was freeze-dried, and the protein concentration of the remaining food in each well was determined using the BCA protein concentration assay to calculate the daily amount of food consumed by the fruit flies.

[0107] 4. Experimental data recording: Under each dietary condition, record the changes in the daily food intake of fruit flies; compare the differences in food intake of fruit flies under different dietary conditions, and explore the relationship between dietary composition and lifespan by recording the changes in the survival rate of fruit flies under each dietary condition.

[0108] 5. Results and Discussion: Compared with a conventional diet, fruit flies on a high-sugar diet showed a significant increase in food intake, possibly due to their preference for high-sugar foods, leading to increased energy intake. However, a prolonged high-sugar diet can trigger metabolic disorders, such as insulin resistance or fat accumulation, thus affecting lifespan and health. The high-protein diet group showed a slight decrease in food intake, possibly because high-protein foods are more difficult to digest or meet the flies' nutritional needs, reducing their total food intake. This diet may lead to excessive protein accumulation, causing physiological stress or toxic reactions, thereby affecting lifespan. The high-fat diet group showed similar food intake to the conventional diet, but due to the high energy density of fat, the flies' total energy intake increased. A long-term high-fat diet may lead to fat accumulation and metabolic imbalance in fruit flies, thus affecting their health and lifespan.

[0109] Figure 5 This is a graph showing the survival rate changes of Oregon-R fruit flies under different dietary conditions, as provided in Example 2 of this application. Figure 5 It can be seen that the survival rate of fruit flies in the high-sugar and high-fat diet groups was significantly lower than that in the conventional diet group, while there was no significant difference in the survival rate of fruit flies in the high-protein diet group and the conventional diet group.

[0110] Figure 6 This is a graph showing the difference in daily food intake of Oregon-R fruit flies under different dietary conditions, as provided in Example 2 of this application. Figure 6 It can be seen that the food intake of fruit flies in the high-sugar diet group increased significantly, the food intake of fruit flies in the high-protein diet group decreased slightly, and the food intake of fruit flies in the high-fat diet group was similar to that of the conventional diet.

[0111] Example 3

[0112] Flavonoids possess various biological activities, including antioxidant and anti-inflammatory effects, and may have different impacts on the feeding behavior and health of fruit flies. This study investigated the effects of adding 36 different flavonoids to a liquid diet of fruit flies on their feeding behavior using a high-throughput feed intake assay.

[0113] Experimental Design:

[0114] 1. Experimental subjects: Male Oregon R fruit flies on the first day of emergence were selected and divided into different treatment groups, each corresponding to a flavonoid compound, for a total of 36 treatment groups. A control group was also set up. Among them, spermidine (1mM) group was the positive control group, with 15 fruit flies in each group.

[0115] 2. Addition of flavonoids: Each flavonoid compound was dissolved in a standard liquid food (the liquid food consisted of 2.5% sucrose, 5% yeast extract, 0.3% propionic acid, 0.1% sodium benzoate, 0.5% Tween 20, and the remainder being water) at a concentration of 10 μM. Flavonoids include quercetin, naringenin, hesperidin, isochoridin, sennaol, hypersennaol, hesperidin / citrus aurantium glycoside, lemon balm glycoside, naringin, naringenin, hesperidin, neohesperidin, sennain, neosennain, robinin, hesperidin, hesperidin, heptamethoxyflavonoids, isosweet orange flavonoids, sweet orange flavonoids, 5,7,8,4'-tetramethoxyflavonoids, 4',5,6,7-tetramethoxyflavonoids, 5-hydroxynorhesperidin, gardenia flavonoid B, phloretin, trifleurone, dihydroquercetin, isoquercetin, rutin, phloretin, trifolin, 3-hydroxyphloretin, 3-hydroxyphloretin, naringin dihydrochalcone, neohesperidin dihydrochalcone, and phloretin-3',5'-di-C-β-glucoside ketone.

[0116] 3. Experimental procedure: Each group of fruit flies independently fed food containing specific flavonoid compounds in a WAFFL. The food supply in each well of the 96-well plate was 20 μl. After feeding for one day, the food intake of each group of fruit flies was accurately measured using the method of this invention.

[0117] 4. Experimental data recording: Record the differences in fruit fly feeding amount under each type of flavonoid compound treatment, compare the effects of 36 types of flavonoid compounds on fruit fly feeding behavior, and analyze the inhibitory or promoting effects of different flavonoids on feeding.

[0118] Figure 7 This is a graph showing the effect of 36 flavonoid compounds provided in Example 3 of this application on the daily food intake of fruit flies. Figure 7 It was found that different flavonoid compounds had varying effects on the food intake of fruit flies. Compared with the control group, some flavonoids, including hesperidin, isochorin, naringin, 5,7,8,4'-tetramethoxyflavonoids, 4',5,6,7-tetramethoxyflavonoids, 5-hydroxynorhesperidin, and genistein B, may have reduced the food intake of fruit flies due to their antioxidant or metabolic regulatory effects. Some flavonoid compounds increased the food intake of fruit flies, including phlorizin, trifolin, and 3-hydroxyphlorizin. The remaining flavonoid compounds did not affect the food intake of fruit flies. When investigating the anti-aging effects of flavonoid compounds on fruit flies, the focus should be on compounds that significantly reduce the food intake of fruit flies. This is because, in biological research, reduced food intake (especially reduced protein intake) is often associated with prolonged lifespan and improved health indicators in experimental models; this phenomenon is known as calorie restriction. In addition, attention should be paid to compounds that do not affect food intake but may exert anti-aging effects through other mechanisms, such as antioxidant, anti-inflammatory, or cell signaling regulation. By screening different flavonoid compounds, this invention can provide data support for the development of flavonoid-based anti-aging and metabolic regulation drugs.

[0119] Furthermore, one or more technical solutions in the embodiments of this application have at least the following technical effects or advantages:

[0120] This application provides a high-precision, high-throughput, and easy-to-operate method for measuring fruit fly food intake. It combines WAFFL (high-throughput fruit fly feeding platform), 96-well cell culture plates, and BCA protein concentration measurement methods. By tracking changes in protein content in liquid food, the change in liquid volume is calculated, thereby accurately measuring the fruit fly's food intake. This solves the problems of feeding behavior interference, liquid evaporation loss, and difficulty in achieving high throughput in existing methods such as dye methods and capillary feeding methods.

[0121] This application provides a high-throughput experimental platform capable of simultaneously measuring multiple samples, and it features flexible experimental design. By adjusting variables such as food composition, drug addition, and environmental conditions, this invention can conduct complex multivariate experiments, significantly improving research efficiency. This platform is particularly suitable for nutritional, drug screening, and behavioral studies requiring large-scale data analysis.

[0122] In this embodiment of the application, considering the shortcomings of traditional methods for measuring fruit fly feed intake in terms of accuracy, throughput, and experimental interference, the present invention aims to provide a high-throughput method for measuring fruit fly feed intake based on BCA protein concentration. This method can accurately measure the feed intake of multiple fruit flies without interfering with their normal physiological behavior, overcoming the problems of evaporation, low throughput, and insufficient accuracy in traditional methods.

[0123] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for determining the amount of food consumed by fruit flies, the method comprising: Each of the multiple fruit flies to be tested is transferred to each hole of the feeding plate of the high-throughput fruit fly feeding platform; Liquid food with a set chemical composition is added into each hole of the food base plate of the high-throughput fruit fly feeding platform; The test fruit flies were allowed to feed freely for a set period of time. The protein content of the remaining liquid food in each well of the food substrate after free feeding was determined to obtain the remaining protein content of each well; as well as Based on the remaining protein content in each well, the corresponding food intake of each fruit fly to be tested was obtained; The specified chemical components include: edible surfactants, wherein the mass of the edible surfactants is 0.1% to 1% of the total mass of the liquid food; The determination of the protein content in the remaining liquid food in each hole of the food substrate includes: Solvent from the remaining liquid food in each hole of the food base plate was removed to obtain multiple powder samples; The protein content of each of the multiple powder samples was determined. The solvent in the remaining liquid food is removed from each hole of the food base plate by freeze drying, wherein the vacuum degree of freeze drying is <10 Pa and the freeze drying time is 18h to 24h; The protein content in the plurality of powder samples was determined using the BCA method or the Bradford method. The amount of food consumed satisfies the following relationship: I=(C0-C1) / C0×V In the formula, I represents the amount of food consumed, C0 represents the protein content of the liquid food before consumption, C1 represents the protein content of the liquid food after consumption, and V represents the volume of the liquid food before consumption.

2. The method according to claim 1, characterized in that, The edible surfactants include one or more of Tween 20, Tween 80, and polyethylene glycol.

3. The method according to claim 2, characterized in that, The specified chemical components also include: sucrose, yeast extract, propionic acid, sodium benzoate, and solvents.

4. The method according to claim 1, characterized in that, The volume of liquid food in each hole of the food base plate is 10μL~30μL.

5. An application of the method according to any one of claims 1 to 4, wherein the method is used to investigate the effects and trends of different dietary or drug treatments on the feeding behavior of fruit flies.

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