High-throughput fruit fly food intake measuring method and application thereof

Abstract

The invention provides a high-throughput fruit fly food intake measuring method and application thereof, and belongs to the field of animal food intake research. The method comprises the following steps: correspondingly transferring each of a plurality of fruit flies to be detected into each hole of a feeding plate of a high-throughput fruit fly feeding platform; respectively adding liquid food with set chemical components into each hole of a food bottom plate of the high-flux fruit fly feeding platform; enabling a plurality of to-be-detected fruit flies to freely ingest at a set time; measuring the content of protein in the residual liquid food after free ingestion to obtain the content of residual protein in each hole; and obtaining the food intake of each corresponding fruit fly to be detected according to the residual protein content in each hole. Based on the combination of a high-throughput fruit fly feeding platform and protein concentration measurement, the change of the liquid volume is calculated by tracking the change of the content of protein in liquid food, so that the fruit fly food intake measurement efficiency and accuracy are improved while the fruit fly food intake measurement is performed in a high-throughput manner.

Description

Technical Field

[0001] The present application relates to the technical field of animal food intake research, and in particular to a high-throughput fruit fly food intake determination method and its application. Background Art

[0002] As one of the classic model organisms, Drosophila melanogaster has been widely used in the fields of nutrient metabolism, drug screening and aging research. The study of Drosophila feeding behavior is of great significance in revealing the relationship between nutrient intake and life processes.

[0003] At present, a variety of methods have been used to study the feeding behavior of fruit flies, which can reflect the feeding characteristics of fruit flies from different levels and angles. The dye method uses dyes in food to mark and infer the food intake of fruit flies by measuring the content of dyes. The radioactive isotope labeling method uses radioactive isotopes to label food and measure the content of isotopes to reflect the food intake. The capillary glass feeding method ( The FLIC method indirectly measures the food intake of fruit flies 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. These methods realize the automated measurement of fruit fly food intake by converting electrical signals. Although the above methods can provide quantitative data to a certain extent, due to the complexity of fruit fly feeding behavior, the measurement accuracy is often affected by factors such as food evaporation and sample loss, and these methods show obvious limitations when processing a large number of samples. Therefore, the development of a method for measuring fruit fly food intake that can simultaneously achieve high throughput, high efficiency and accuracy has become an urgent need in current research. Summary of the invention

[0004] The present application provides a high-throughput fruit fly food intake measurement method and application thereof to solve the following technical problem: how to improve the efficiency and accuracy of fruit fly food intake measurement while measuring fruit fly food intake in a high-throughput manner.

[0005] In a first aspect, the present application provides a method for determining the food intake of fruit flies, the method comprising:

[0006] Transferring each of the multiple fruit flies to be tested to each well of a feeding plate of a high-throughput fruit fly feeding platform;

[0007] Adding liquid food with set chemical composition into each well of the food bottom plate of the high-throughput fruit fly feeding platform;

[0008] At a set time, allowing the plurality of fruit flies to be tested to eat freely;

[0009] Determining the protein content of the remaining liquid food in each well of the food bottom plate after free feeding to obtain the remaining protein content in each well; and

[0010] According to the residual protein content in each well, the corresponding food intake of each fruit fly to be tested is obtained.

[0011] Optionally, the set chemical composition includes: edible surfactant.

[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 set chemical composition also includes: sucrose, yeast extract, propionic acid, sodium benzoate and solvent.

[0014] Optionally, the step of determining the protein content of the remaining liquid food in each hole of the food base plate comprises:

[0015] removing the solvent in the remaining liquid food in each well of the food bottom plate to obtain a plurality of powder samples;

[0016] The protein contents in the plurality of powder samples are respectively determined.

[0017] Optionally, the removal of the solvent in the remaining liquid food in each hole of the food bottom plate is performed by freeze drying, the vacuum degree of the freeze drying is less than 10 Pa, and the freeze drying time is 18 hours to 24 hours.

[0018] Optionally, the protein contents in the multiple powder samples are determined respectively using a BCA method or a Bradford method.

[0019] Optionally, the food intake satisfies the following relationship:

[0020] I=(C0-C1) / C0×V

[0021] Wherein, I represents the food intake, C0 represents the protein content in the liquid food before ingestion, C1 represents the protein content in the liquid food after ingestion, and V represents the volume of the liquid food before ingestion.

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

[0023] In a second aspect, the present application provides an application of the method described in any one of the embodiments of the first aspect, wherein the method is used to explore the effects of the addition of different nutrients or drugs on the feeding behavior of fruit flies.

[0024] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0025] The present application provides a method for determining the food intake of fruit flies, the method comprising: transferring each of a plurality of fruit flies to be tested to each hole of a feeding plate of a high-throughput fruit fly feeding platform; adding liquid food with set chemical composition to each hole of a food bottom plate of the high-throughput fruit fly feeding platform; allowing the plurality of fruit flies to be tested to freely ingest food at a set time; determining the protein content in the remaining liquid food in each hole of the food bottom plate after the free ingestion to obtain the remaining protein content in each hole; and obtaining the corresponding food intake of each fruit fly to be tested based on the remaining protein content in each hole. The present application is based on a combination of a high-throughput fruit fly feeding platform, a porous food bottom plate and a protein concentration determination, and calculates the change in liquid volume by tracking the change in the protein content in the liquid food, thereby improving the efficiency and accuracy of the fruit fly food intake determination while performing a high-throughput determination of the fruit fly food intake. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0027] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0028] Figure 1 A flow chart of a method for measuring the food intake of fruit flies provided in an embodiment of the present application;

[0029] Figure 2 The high-throughput fruit fly feeding platform provided in Example 1 of the present application;

[0030] Figure 3 A schematic diagram of the method for measuring the food intake of fruit flies provided in Example 1 of the present application;

[0031] Figure 4 This is a diagram for verifying the actual application effect of the fruit fly food intake provided in Example 1 of the present application;

[0032] Figure 5 A graph showing the changes in the survival rate of Oregon-R fruit flies under different dietary conditions provided in Example 2 of the present application;

[0033] Figure 6This is a graph showing the difference in food intake of Oregon-R fruit flies in one day under different dietary conditions provided in Example 2 of the present application;

[0034] Figure 7 This is a graph showing the effects of the 36 flavonoid compounds provided in Example 3 of the present application on the daily food intake of fruit flies. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0036] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have 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., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0037] In addition, in the description of the specification of the present application, the terms "including", "comprising", etc. mean "including but not limited to". In this article, relational terms such as "first" and "second", etc. are only used 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 article, "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A and B can be singular or plural. In this article, "at least one" means one or more, and "plurality" means two or more. "At least one", "the following at least one item (items)" or similar expressions refer to any combination of these items, including any combination of single items (items) or plural items (items). For example, "at least one of a, b, or c", or "at least one of a, b, and c", can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple. "Parts" such as parts by weight and parts by mass indicate the proportional relationship between the components. In the proportional relationship involved in this article, the parameters that need to be described by proportion should be understood as the first term of the proportional formula in the order of description, and the proportional numbers should be understood as the second term of the proportional formula. For example, the mass ratio of substance A, substance B, and substance C is 1:2:3, then substance A, substance B, and substance C should correspond one-to-one with the proportional numbers in the proportional formula in the order of description, that is, the mass of substance A: the mass of substance B: the mass of substance C = 1:2:3.

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

[0039] Figure 1 A schematic flow chart of a method for measuring food intake of fruit flies provided in an embodiment of the present application.

[0040] The present application provides a method for determining the food intake of fruit flies, such as Figure 1 As shown, the method includes:

[0041] S1. Transferring each of the multiple fruit flies to be tested to each well of a feeding plate of a high-throughput fruit fly feeding platform;

[0042] The high-throughput fruit fly feeding platform achieves precise control of the fruit fly feeding environment through an integrated design, including temperature, humidity, and light conditions, which are key factors affecting the growth and development of fruit flies. More importantly, the platform supports large-scale, multivariate experimental design, and can support experimental design with multiple experimental variables (such as the addition of different nutrients or drugs) at the same time, which is suitable for large-scale screening experiments.

[0043] The feeding plate is a component of a high-throughput fruit fly feeding platform for placing fruit flies, and usually has a plurality of small holes or channels, each of which can accommodate one or more fruit flies.

[0044] S2, adding liquid food with set chemical composition into each well of the food bottom plate of the high-throughput fruit fly feeding platform;

[0045] It should be noted that, in the embodiments of the present application, the food base plate can be a common 96-well plate for placing and providing liquid food required by fruit flies. In the high-throughput fruit fly feeding platform, the positions of the holes on the feeding plate match the 96-well plate to ensure that the food can be evenly distributed to the position of each fruit fly.

[0046] Steps S1 and S2 ensure that each fruit fly has an independent feeding space and avoid mutual influence between fruit flies.

[0047] In some embodiments, the set chemical composition includes: an edible surfactant.

[0048] Optimizing the liquid food formula is an important factor in ensuring the normal feeding behavior of fruit flies. The chemical composition of the liquid food can be set according to the experimental requirements to study the effects of different ingredients on the food intake of fruit flies. The present application adds edible surfactants to the liquid food, which effectively reduces the viscosity of the liquid food. The innovations brought about by this measure include: ① Improving the feeding behavior of fruit flies: reducing viscosity makes the fruit flies feed more smoothly and avoids feeding difficulties caused by excessive viscosity, thereby improving the naturalness of the feeding behavior and the accuracy of the data. ② Experimental repeatability and accuracy: The addition of edible surfactants can prevent excessive adhesion of liquid food to the feeding plate, ensure the consistency of food volume and food intake measurements in each experiment, and improve the repeatability and accuracy of the experiment.

[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 food. At the same time, the mass of edible surfactant is limited to 0.1% to 1% of the total mass of the liquid food, which has no significant effect on the health of fruit flies and is safe for consumption. Exemplarily, the mass of edible surfactant can be 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1% and the like of the total mass of the liquid food.

[0051] In some embodiments, the set chemical composition further includes: sucrose, yeast extract, propionic acid, sodium benzoate and solvent.

[0052] Sucrose is the main carbon source in fruit fly liquid food and provides energy for fruit flies.

[0053] Yeast extract is an important nitrogen source in Drosophila liquid food, providing Drosophila with essential amino acids and other nutrients.

[0054] Propionic acid is mainly used as a pH buffer in fruit fly liquid food. Fruit flies produce carbon dioxide during feeding, which causes the food to become acidic. Propionic acid can adjust the pH value of the food and keep it within the range suitable for fruit flies.

[0055] Sodium benzoate is mainly used as a preservative in fruit fly liquid food. It can inhibit the growth of bacteria and extend the shelf life of food.

[0056] S3, allowing the plurality of fruit flies to be tested to freely ingest food at a set time;

[0057] S4, measuring the protein content of the remaining liquid food in each well of the food bottom plate after free feeding to obtain the remaining protein content in each well;

[0058] In some embodiments, the step of determining the protein content of the remaining liquid food in each well of the food base plate comprises:

[0059] removing the solvent in the remaining liquid food in each well of the food bottom plate to obtain a plurality of powder samples;

[0060] The protein contents in the plurality of powder samples are respectively determined.

[0061] The present application calculates the change in liquid volume by tracking the change in protein content in liquid food, thereby accurately measuring the food intake of fruit flies, solving the problems of feeding behavior interference, liquid evaporation loss and difficulty in achieving high throughput that exist in existing measurement methods such as the dye method and capillary feeding method.

[0062] In some embodiments, the removal of the solvent in the remaining liquid food in each hole of the food bottom plate is performed by freeze drying, the vacuum degree of the freeze drying is less than 10 Pa, and the freeze drying time is 18 hours to 24 hours.

[0063] The freeze drying technology is used to remove the residual water in the food bottom plate after the fruit flies have eaten, thereby avoiding the difference in liquid volume in each hole caused by the difference in feeding behavior between individual fruit flies. Compared with the traditional air-blast drying method, it has the advantages of easy dissolution and rehydration, and prolonged shelf life. Exemplarily, the freeze drying time can be 18h, 19h, 20h, 21h, 22h, 23h, 24h, etc.

[0064] In some embodiments, the protein contents in the plurality of powder samples are determined respectively using a BCA method or a Bradford method.

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

[0066] The food intake of fruit flies is estimated by measuring the residual protein content in liquid food, which has the following characteristics: ① Avoid dye interference: BCA method and Bradford method do not need to add additional dyes to the food, avoiding the potential interference of dyes on the feeding behavior of fruit flies. ② High sensitivity and accuracy: BCA method and Bradford method can detect extremely low concentrations of protein content, and the accuracy and sensitivity are significantly better than the dye method, ensuring the accuracy of food intake measurement. ③ Data consistency: This method can accurately measure the feeding differences 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: obtaining the corresponding food intake of each fruit fly to be tested according to the residual protein content in each well.

[0068] In some embodiments, the food intake satisfies the following relationship:

[0069] I=(C0-C1) / C0×V

[0070] Wherein, I represents the food intake, C0 represents the protein content in the liquid food before ingestion, C1 represents the protein content in the liquid food after ingestion, and V represents the volume of the liquid food before ingestion.

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

[0072] Analysis methods of fruit fly food intake results: ① Accurate determination of food intake: Accurate determination of food intake can be analyzed by statistical software (Graphpad Prism 9) to compare the differences in food intake of fruit flies 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: In order 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 food intake between different treatment groups.

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

[0074] 1. High throughput and high efficiency: The present invention uses a high-throughput fruit fly feeding platform to process multiple fruit flies at the same time, significantly improving the throughput and efficiency of the experiment. This large-scale, synchronous processing capability is particularly suitable for experiments that require a large amount of data, such as nutrition research and drug screening. Compared with the traditional single fruit fly feeding method, the platform can obtain a large amount of data in a short time, effectively reducing the experimental cycle and labor intensity.

[0075] 2. High-precision food intake measurement: Through the protein concentration measurement method, the present invention can avoid the interference problem that the traditional dye method may cause to the fruit fly's feeding behavior. The dye method may affect the fruit fly's feeding preference due to the need to add additional chemicals, resulting in data distortion. The protein content in the liquid food is not affected by evaporation during the experiment. Compared with the dye or capillary method, the present invention uses a standard curve to accurately calculate the fruit fly's food intake, eliminating the interference of external factors on the food intake measurement, and ensuring the stability and accuracy of the data.

[0076] 3. Optimization of liquid food formula: The present invention adds surfactants to liquid food, which effectively reduces the viscosity of liquid food and prevents the adverse effects of high food viscosity on the feeding and survival of fruit flies, such as difficulty in feeding or accidental ingestion of residual food on the feeding plate. It can also prevent high-viscosity liquid from excessively adhering to the feeding plate and affecting the accuracy of food intake measurement, further improving 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 research. By accurately measuring the feeding behavior and its changing trend of fruit flies under different diets or drug treatments, the effects of different ingredients on fruit fly metabolism, lifespan and health can be effectively analyzed. This precise data support helps to better understand the mechanism of action of nutrients and drugs, and provides a reference model for human health research.

[0078] Based on a general inventive concept, the present application provides an application of the method described in any one of the above embodiments, wherein the method is used to explore the effects of the addition of different nutrients or drugs on the feeding behavior of fruit flies.

[0079] By utilizing a high-throughput fruit fly feeding platform (WAFFL) and a protein concentration detection method, the present application can simultaneously measure fruit fly food 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 will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to industry standards. If there is no corresponding industry standard, then the 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 that 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: The standardized 96-well plate is used to process multiple fruit fly samples at the same time, and each well is only for a single fruit fly to ingest.

[0084] 3. Freeze dryer: used to remove moisture from liquid food after fruit flies have eaten, to avoid interference of feeding differences between individual fruit flies on the results of food intake measurement, and to improve the accuracy of the experiment and data reliability.

[0085] 4. Liquid food for fruit flies: including 2.5% sucrose, 5% yeast extract, 0.3% propionic acid, 0.1% sodium benzoate, 0.5% Tween 20, and the balance is water.

[0086] 5. BCA protein concentration test kit: used to determine the protein concentration in liquid food before and after ingestion.

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

[0088] Example 1

[0089] This embodiment provides a method for determining the food intake of fruit flies, which combines WAFFL (high-throughput fruit fly feeding platform), 96-well cell culture plates and BCA protein concentration determination, and the method specifically includes the following steps:

[0090] Step 1, liquid food preparation: Mix the ingredients in the liquid food formula evenly, and make sure there are no obvious particles or insoluble substances in the solution. Use a filter to filter the food to ensure its cleanliness and stability. The composition of the liquid food can be flexibly adjusted according to the experimental needs, such as by adding specific drugs or nutrients to test their effects on the feeding behavior of fruit flies.

[0091] Step 2. Preparation of fruit flies: Use healthy and uniform fruit fly strains for experiments, usually wild-type fruit flies (OregonR) or other fruit fly strains commonly used in laboratories. Group the fruit flies according to sex, age, and weight to ensure data consistency and experimental comparability.

[0092] Step 3, feeding fruit flies: Use WAFFL to feed fruit flies. Each well of the 96-well food plate is only used for the corresponding single fruit fly in the feeding plate to prevent interference between fruit flies. Add 20 μl of liquid food to each well to ensure that the liquid is evenly distributed at the bottom of the well. According to the specific experimental design, fruit flies can freely eat in the chamber for a certain period of time (such as 24 hours).

[0093] Step 4, remove the residual moisture in the well plate after the fruit flies have ingested: After the prescribed feeding time is over, slowly remove the 96-well plate and put it into a -80 refrigerator for precooling (1-2h). After the vacuum degree of the freeze dryer reaches below 10Pa, continue to run it for 18-24 hours until the sample in the well plate is in powder form.

[0094] Step 5, BCA protein concentration determination: add 100 μl of PBS (diluted 5 times) to each well of the freeze-dried 96-well plate, use the BCA protein concentration detection kit to prepare the standard solution and BCA working solution, take 10 μl of the diluted sample in each well to another 96-well plate, add 100 μl of BCA working solution respectively, mix well, place at 37°C for 30 minutes, detect the absorbance at 562 nm by an enzyme reader, and determine the absorbance of each well.

[0095] Step 6. Calculation of fruit fly food intake: Draw a standard curve based on a protein standard of known concentration. Compare the absorbance changes of each well before and after the fruit fly ingests food with the standard curve to obtain the protein concentration of each well, and then calculate the food intake of each fruit fly in a specific time based on the ratio of protein amount to liquid volume.

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

[0097] ② Fruit fly food intake (μl) = [protein content before feeding (μg) - remaining protein content (μg)] / protein content before feeding (μg) × original liquid food volume (μl).

[0098] Figure 2 The components of the high-throughput fruit fly feeding platform provided in Example 1 of the present application. Figure 2 Figure 2. (a) WAFFL feeding plate. (b) Food is located in a 96-well plate underneath the WAFFL, allowing up to 96 parallel experiments to be performed in a single WAFFL. The top is sealed with a reusable silicone mat to maintain humidity. (c) To visualize the behavior of the fruit flies during the experiment, the silicone mat can be replaced with an acrylic cover. (d) To transfer the fruit flies while maintaining their position on the 96-well plate, a transfer adapter and receiving plate are used.

[0099] Figure 3 This is a schematic diagram of the method for measuring the food intake of fruit flies provided in Example 1 of the present application. Figure 3 It can be seen that based on the WAFFL platform, 96-well cell culture plates and BCA protein concentration assay, the food intake of fruit flies in one day can be measured.

[0100] Figure 4 This is a diagram for verifying the actual application effect of the fruit fly food intake provided in Example 1 of the present application; Figure 4 In this paper, (a) the evaporation of liquid food does not affect the protein content in the food; (b) the volume of liquid food measured by this method is proportional to the protein content, and the correlation coefficient R 2 Reaching 0.9973; (c) accurately measuring the compensatory feeding of fruit flies to different food concentrations; (d) accurately measuring the food intake of fruit flies of different sexes and mating states.

[0101] Example 2

[0102] This example studies the effects of different dietary ingredients on the feeding behavior, metabolism and health of fruit flies by accurately measuring the food intake of fruit flies under different dietary conditions, providing data support for understanding the mechanism of action of nutrition on organisms.

[0103] Experimental design:

[0104] 1. Experimental subjects: Male OregonR fruit flies on the first day of eclosion were selected and divided into three groups, each containing 96 fruit flies.

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

[0106] 3. Experimental process: In the WAFFL system, 20 μl of liquid food (corresponding to different diet formulas) was added to each well of the 96-well plate, and only one fruit fly was placed in each well to ensure independent feeding. Fruit flies were allowed to feed freely in their respective chambers, and their food intake was monitored regularly. From the first to the seventh day of the experiment, changes in food intake were recorded every day. After each day of 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 food intake of the fruit flies.

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

[0108] 5. Results and discussion: Compared with the conventional diet, the high-sugar diet significantly increased the food intake of fruit flies, which may be due to the preference of fruit flies for high-sugar foods, resulting in increased energy intake. However, a long-term high-sugar diet can induce metabolic disorders, such as insulin resistance or fat accumulation, which in turn affects lifespan and health status; the food intake of fruit flies in the high-protein diet group decreased slightly, which may be because high-protein foods are difficult to digest or meet the nutritional needs of fruit flies, reducing the total food intake of fruit flies. This diet may lead to excessive accumulation of protein, causing physiological stress or toxic reactions, thereby affecting lifespan; the food intake of fruit flies in the high-fat diet group was similar to that of the conventional diet, but due to the high energy density of fat, the total energy intake of fruit flies increased. Long-term high-fat diet may lead to fat accumulation and metabolic imbalance in fruit flies, thus affecting health and lifespan.

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

[0110] Figure 6 This is a graph showing the difference in food intake of Oregon-R fruit flies in one day under different dietary conditions 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 regular diet.

[0111] Example 3

[0112] Flavonoid compounds have multiple biological activities such as anti-oxidation and anti-inflammatory, and may have different effects on the feeding behavior and health of fruit flies. This example uses a high-throughput feeding amount measurement method to explore the effects of 36 different flavonoid compounds added to the liquid food of fruit flies on their feeding behavior.

[0113] Experimental design:

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

[0115] 2. Addition of flavonoid compounds: Each flavonoid compound was dissolved in a standard liquid food (the ingredients of the liquid food were: 2.5% sucrose, 5% yeast extract, 0.3% propionic acid, 0.1% sodium benzoate, 0.5% Tween 20, and the remainder was water) and the concentration was set to 10 μM. Flavonoid compounds include quercetin, naringenin, hesperidin, isosakuratin, eriodictyol, homoeriodictyol, aurantium glycosides / citrus aurantium, balsam pear glycosides, naringin, naringin, hesperidin, neohesperidin, eriodictyol, neoeriodictyol, wild sumac glycosides, nobiletin, tangeretin, heptamethoxyflavone, iso-sweet aurantium flavone, sweet aurantium flavone, 5,7,8,4'-tetramethoxyflavone, 4',5,6,7-tetramethoxyflavone, 5-hydroxynornobiletin, gardeniaflavin B, phloretin, trilobate caryophyllidin, dihydroquercetin, isoquercetin, rutin, phloretin, trilobate glycosides, 3-hydroxyphloretin, 3-hydroxyphloretin, naringin dihydrochalcone, neohesperidin dihydrochalcone, phloretin-3',5'-di-C-β-glucoside ketone.

[0116] 3. Experimental process: Each group of fruit flies is fed with food containing specific flavonoid compounds independently in WAFFL. The food supply volume in each well of the 96-well plate is 20 μl. After the fruit flies have been fed for one day, the food intake of each group of fruit flies is accurately measured using the method of the present invention.

[0117] 4. Experimental data recording: record the difference in fruit fly food intake under each flavonoid treatment, compare the effects of 36 flavonoids on fruit fly feeding behavior, and analyze the effects of different flavonoids in inhibiting or promoting feeding.

[0118] Figure 7 This is a graph showing the effects of 36 flavonoid compounds provided in Example 3 of the present application on the daily food intake of fruit flies. Figure 7 It can be seen that different flavonoid treatments have different effects on the food intake of fruit flies. Compared with the control group, some flavonoids may reduce the food intake of fruit flies due to antioxidant or metabolic regulation effects, including hesperidin, isosakura glycosides, naringin, 5,7,8,4'-tetramethoxyflavone, 4',5,6,7-tetramethoxyflavone, 5-hydroxynornobiletin, and gardeniaflavone B. Some flavonoid compounds increase the food intake of fruit flies, including phloretin, trilobatin, and 3-hydroxyphloretin. The remaining flavonoid compounds do not affect the food intake of fruit flies. When exploring the anti-aging effects of flavonoid compounds on fruit flies, we should focus on compounds that significantly reduce the food intake of fruit flies. This is because in biological studies, reducing food intake (especially reducing protein intake) is often associated with extending the lifespan of experimental models and improving health indicators. This phenomenon is called calorie restriction. In addition, attention should also be paid to compounds that do not affect food intake but may exert anti-aging effects through other mechanisms. These compounds may exert anti-aging effects through antioxidant effects, anti-inflammatory effects, or regulation of cell signal transduction. By screening different flavonoid compounds, the present invention can provide data support for the development of anti-aging and metabolic regulation drugs based on flavonoids.

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

[0120] In the embodiments of the present application, a high-precision, high-throughput and easy-to-operate method for measuring the food intake of fruit flies is provided, which combines WAFFL (high-throughput fruit fly feeding platform), 96-well cell culture plates and BCA protein concentration determination method, and accurately measures the food intake of fruit flies by tracking the changes in the protein content in liquid food to infer the changes in liquid volume, thereby solving the problems of feeding behavior interference, liquid evaporation loss and difficulty in achieving high throughput in existing determination methods such as dye method and capillary feeding method.

[0121] In the embodiments of the present application, a high-throughput experimental platform is provided that can measure multiple samples simultaneously and has flexible experimental design. By adjusting variables such as food ingredients, drug additions, and environmental conditions, the present invention can conduct complex multivariate experiments and significantly improve research efficiency. The platform is particularly suitable for nutrition, drug screening, and behavioral research that requires large-scale data analysis.

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

[0123] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A method for measuring the food intake of fruit flies, the method comprising: Transferring each of the multiple fruit flies to be tested to each well of a feeding plate of a high-throughput fruit fly feeding platform; Adding liquid food with set chemical composition into each well of the food bottom plate of the high-throughput fruit fly feeding platform; At a set time, allowing the plurality of fruit flies to be tested to eat freely; Determining the protein content of the remaining liquid food in each well of the food bottom plate after free feeding to obtain the remaining protein content in each well; as well as According to the residual protein content in each well, the corresponding food intake of each fruit fly to be tested is obtained.

2. The method according to claim 1, characterized in that The set chemical components include: edible surfactant.

3. The method according to claim 2, characterized in that 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.

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

5. The method according to claim 1, characterized in that: The step of determining the protein content of the remaining liquid food in each hole of the food bottom plate comprises: removing the solvent in the remaining liquid food in each well of the food bottom plate to obtain a plurality of powder samples; The protein contents in the plurality of powder samples are respectively determined.

6. The method according to claim 5, characterized in that The method for removing the solvent from the remaining liquid food in each hole of the food bottom plate uses freeze drying, the vacuum degree of the freeze drying is less than 10 Pa, and the freeze drying time is 18 hours to 24 hours.

7. The method according to claim 5, characterized in that The protein contents in the plurality of powder samples are respectively determined by using the BCA method or the Bradford method.

8. The method according to claim 1, characterized in that: The food intake satisfies the following relationship: I=(C0-C1) / C0×V Wherein, I represents the food intake, C0 represents the protein content in the liquid food before ingestion, C1 represents the protein content in the liquid food after ingestion, and V represents the volume of the liquid food before ingestion.

9. The method according to claim 1, characterized in that: The volume of the liquid food in each well of the food bottom plate is 10 μL to 30 μL.

10. Use of the method according to any one of claims 1 to 9, wherein the method is used to explore the effects and changing trends of different diets or drug treatments on the feeding behavior of fruit flies.

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