Method for determining binary feeding preference of drosophila melanogaster based on single-color marking of sunset yellow dye

Abstract

According to the method for measuring the feeding preference of the drosophila melanogaster based on the single-color marker of the sunset yellow dye, the two kinds of food are separately marked by 0.15% of the sunset yellow dye in two sets of feeding preference measuring tubes respectively, and the sunset yellow dye in the food can not be absorbed by the drosophila melanogaster, so that the sunset yellow dye can be completely discharged out of the body of the drosophila melanogaster. The sunset yellow dye in excrement of the drosophila melanogaster after eating is collected and dissolved in a buffer solution, and the feeding preference of the drosophila melanogaster to two kinds of food can be calculated by measuring the absorbance of the solution.

Description

Technical Field

[0001] The invention belongs to the field of fruit fly feeding preference detection methods, and specifically relates to a method for measuring fruit fly binary feeding preference based on sunset yellow dye monochromatic marking. Background Art

[0002] Drosophila is a classic model animal in animal behavior research, especially used to study the regulatory mechanism of feeding behavior. The determination of Drosophila binary feeding preference is an important experimental content in feeding behavior research, and is often used as a basic indicator to reflect the effects of nutrition, drugs and other intervention factors on its feeding choice.

[0003] In order to determine the binary feeding preference of fruit flies, a variety of methods have been developed in the prior art, among which the most widely used mainstream methods include: two-color dye labeling method, radioisotope labeling feeding preference determination method, double capillary method and flyPAD method based on electrical signal recording. Among them, the two-color dye labeling method is to mark two foods in the same test tube or culture dish with two different colors of dyes (such as brilliant blue and sulforhodamine B, brilliant blue and orange yellow 4), and after the fruit flies eat, the feeding preference is calculated by observing the color of the fruit fly abdomen or measuring the absorbance of the excrement solution at two wavelengths; the biggest disadvantage of this method is that the fruit flies have different feeding preferences for the two dyes themselves, which will interfere with the measurement results. This problem is particularly serious under the feeding conditions of culture media with complex nutritional composition. The radioisotope labeling feeding preference determination method is to separately mark one of the two foods with the same radioisotope in two groups of fruit fly tubes, and after the fruit flies eat, the fruit flies are killed and their feeding preference is calculated by measuring the radioactivity intensity of the fruit flies. The drawback of this method is that the fruit flies need to be killed, and it is impossible to continuously measure the feeding preference of the same batch of test fruit flies at multiple time points. The double capillary method is to install two sets of glass capillaries in the same fruit fly tube, each set of capillaries contains a liquid culture medium, and the feeding preference of fruit flies is calculated by measuring the drop in the liquid level of the culture medium in the two sets of capillaries. The limitation of this method is that it is only applicable to liquid culture medium, not to the most widely used solid fruit fly culture medium. The flyPAD method calculates the feeding preference of fruit flies by recording the electrical signals generated when two solid culture media placed on electrodes are eaten by fruit flies. The limitation of this method is that it can only record the number of contacts between fruit flies and food, but cannot truly measure the volume or weight of food intake, and it is only applicable to solid culture media with very small surface area (about 1.5mm in diameter), which causes the culture medium to quickly lose water and dry out. Therefore, it is only applicable to feeding preference determination in a short period of time (usually no more than 1 hour), which greatly limits its use scenario.

[0004] In view of the limitations of previous methods for measuring fruit fly feeding preference, we developed a method for measuring fruit fly binary feeding preference based on single-color marking with sunset yellow dye. The advantage of this method is that it uses sunset yellow, a food dye that cannot be absorbed by fruit flies, to separately mark two solid culture media in two sets of feeding preference measurement tubes. This avoids the preference interference caused by multiple dyes. At the same time, since fruit flies do not need to be killed during the entire test process, it is also suitable for long-term and multi-time point continuous measurement of fruit fly feeding preference. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides a method for determining the feeding preference of fruit flies based on single-color marking of sunset yellow dye, which is achieved by the following technical means:

[0006] The present invention discloses a method for determining the feeding preference of fruit flies based on a single-color marker of sunset yellow dye, comprising:

[0007] (1) Two sets of feeding preference test tubes were used to separately label the two foods with 0.15% sunset yellow dye;

[0008] (2) The fruit flies were kept in a feeding preference test tube for 24 hours, and then the sunset yellow dye in the excrement of the fruit flies after feeding was collected and dissolved in a buffer solution. The absorbance of the solution was measured to calculate the feeding preference of the fruit flies for the two foods.

[0009] Furthermore, the step (1) of separately marking the two foods includes:

[0010] Prepare two culture media, A and B, add sunset yellow dye to half of each culture medium, and then distribute them into food cups, wherein food A without dye is paired with food B containing sunset yellow, and food B without dye is paired with food A containing sunset yellow.

[0011] Furthermore, the buffer solution in step (2) is 2 mL of PBST solution.

[0012] Furthermore, the calculation of the fruit fly's feeding preference for the two foods in step (2) includes:

[0013] (2.1) Take 100 μL of each excrement dye solution, use a PBST solution without dye as a blank control, measure the absorbance of the solution at a wavelength of 482 nm using an ELISA reader, and subtract the absorbance of the blank control to obtain the absorbance A and absorbance B of the two excrement dye solutions A and B;

[0014] (2.2) Weigh 10 mg of sunset yellow dye and dissolve it in another 10 ml of dye-free PBST solution to obtain a sunset yellow standard stock solution. Then, the sunset yellow standard stock solution is further diluted with dye-free PBST to multiple concentrations such as 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the original concentration, and the dye-free PBST is used as a blank control. 100 μL of each concentration solution is taken and the absorbance of the sunset yellow standard solution of different concentrations and the blank PBST solution at a wavelength of 482 nm is measured by an ELISA instrument. According to the absorbance and the known dye concentration of the mother solution, a standard curve is calculated and drawn;

[0015] (2.3) Based on the measured absorbance and the calculated standard curve, calculate the average food intake of each fruit fly on culture media A and B;

[0016] (2.4) Based on the calculated amount of food intake by fruit flies for foods A and B, calculate the preference index of fruit flies for the two foods.

[0017] Furthermore, the calculation formula in step (2.3) is:

[0018] Feed intake A = [(0.1028 × absorbance A × 2) / C × 1000] / n (μL);

[0019] Feed intake B = [(0.1028 × absorbance B × 2) / C × 1000] / n (μL);

[0020] Where C is the concentration of sunset yellow dye in the culture medium, and n is the number of fruit flies in each excrement assay tube.

[0021] Furthermore, the calculation formula of the preference index of fruit flies for food A in step (2.4) is:

[0022] Preference index A = (feed intake A - feed intake B) / (feed intake A + feed intake B);

[0023] The calculation formula of the fruit fly's preference index for food B is:

[0024] Preference index B = (feed intake B - feed intake A) / (feed intake A + feed intake B).

[0025] The beneficial effects of the present invention are:

[0026] The advantage of this method is that it uses a food pigment sunset yellow that cannot be absorbed by fruit flies, and separately marks two solid culture media in two sets of feeding preference test tubes. This avoids the preference interference caused by multiple dyes. At the same time, since the fruit flies do not need to be killed during the entire test process, it is also suitable for long-term and multi-time point continuous measurement of fruit fly feeding preferences. In addition, the core device of the present invention has a simple structure, low production cost and is easy to operate, so it has great application prospects in the field of behavioral research. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the feeding preference measurement tube, where:

[0028] (a) The feeding preference test tube consists of three types of parts, namely a round-bottom test tube (1), a test tube cover with a hole (2) and two food cups (3);

[0029] (b) The assembled feeding preference test tube;

[0030] (c) The round bottom test tube has a length of 90 mm, a width of 30.5 mm and a thickness of 1 mm;

[0031] (df) The test tube cover is designed with two large circular holes with a diameter of 9 mm for inserting the food cup (4) and 16 small circular holes with a diameter of 0.8 mm for ventilation (5). The outer diameter of the side wall of the test tube cover (6) is 28.5 mm, the inner diameter is 26.5 mm, the thickness is 1 mm, and the height is 7.5 mm. The bottom diameter of the test tube cover is 31.5 mm and the thickness is 2 mm;

[0032] (g) The outer diameter of the food cup wall is 9 mm, the inner diameter is 8 mm, the height is 10 mm, the base diameter is 11 mm, and the base thickness is 0.5 mm.

[0033] Figure 2 This is the standard curve of sunset yellow dye.

[0034] Figure 3 A detailed procedure for measuring the binary feeding preference of Drosophila using the feeding preference assay tube is provided, in which:

[0035] (a) Prepare two culture media, A and B, add sunset yellow dye to half of each culture medium, and then dispense into food cups;

[0036] (b) Insert the food cup into the large hole on the test tube lid. Pair food A without dye with food B containing sunset yellow, and food B without dye with food A containing sunset yellow. Then transfer the test fruit flies into the test tube and cover the test tube lid with the food cup.

[0037] (c) The fruit flies were kept in a feeding preference test tube for 24 hours. Since the sunset yellow in the food could not be absorbed by the fruit flies, it would be excreted on the tube wall by the fruit flies.

[0038] (d) After 24 hours, the fruit flies were removed from the feeding preference test tubes, and the food cups on the test tube lids were removed. Then, 2 mL of PBST solution was added to each set of feeding preference test tubes. The test tube lids were sealed with tape, and the feeding preference test tubes were shaken to dissolve the sunset yellow dye in the excrement in the PBST solution. The absorbance of the solution was measured by an ELISA instrument, and the fruit fly's feeding preference for the two foods was calculated based on the absorbance.

[0039] Figure 4 The figure shows the amount of food consumed by Drosophila on the complete nutrient medium and the medium without amino acids, sucrose or vitamins under binary selective feeding conditions (left figure). Each dot represents the data of one excrement measurement tube. The feeding preference index of Drosophila for amino acids, sucrose and vitamins (right figure). ****p<0.0001; t test.

[0040] Figure 5 The figure below shows the amount of food consumed by Drosophila on sucrose medium with different concentrations under binary selective feeding conditions (left). Each dot represents the data of one excrement measurement tube. The feeding preference index of Drosophila on 50 mM sucrose medium (right). ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; t-test. DETAILED DESCRIPTION

[0041] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but it should be understood that the embodiments and drawings are only used to exemplify the present invention and do not constitute any limitation on the protection scope of the present invention. All reasonable changes and combinations within the scope of the inventive concept of the present invention fall within the protection scope of the present invention.

[0042] Example 1

[0043] (1) Prepare two experimental culture media A and B. Divide the two culture media A and B into two parts, add 0.15% sunset yellow dye (CAS registration number: 2783-94-0) to one part (i.e., add 0.15g sunset yellow per 1mL culture medium) and mix well. Dispense the obtained four culture media into food cups ( Figure 3 a), and it can be used after it is cooled and solidified. In this example, the two culture media A and B are agar culture media containing 100mM and 20mM amino acids respectively.

[0044] (2) Insert the food cup into the 9 mm large hole on the test tube cover. Pair the food A without dye and the food B containing sunset yellow and install them in the same food cup. Pair the food B without dye and the food A containing sunset yellow and install them in the same food cup. Figure 3 b)

[0045] (3) Transfer the test fruit flies into a test tube and cover the tube with a lid equipped with a food cup.

[0046] (4) The fruit flies were kept in a feeding preference test tube for 24 hours. Since the sunset yellow in the food could not be absorbed by the fruit flies, it was excreted on the tube wall by the fruit flies. Figure 3 c)

[0047] (5) After 24 hours, remove the fruit flies from the feeding preference test tubes, remove the food cups on the test tube lids, and then add 2 mL of PBST solution (phosphate buffered saline containing 0.1% Tween-20) to each set of feeding preference test tubes. Seal the test tube lids with tape, and then shake the feeding preference test tubes to dissolve the sunset yellow dye in the excrement in the PBST solution. Figure 3 d)

[0048] (6) Take 100 μL of each excrement dye solution, use a PBST solution without dye as a blank control, measure the absorbance of the solution at a wavelength of 482 nm by an ELISA instrument, and subtract the absorbance of the blank control. Obtain the absorbance A and absorbance B of the two excrement dye solutions A and B. In this example, the absorbance A after subtracting the blank was measured to be 0.232, and the absorbance B after subtracting the blank was measured to be 0.059.

[0049] (7) Weigh 10 mg of sunset yellow dye and dissolve it in another 10 ml of PBST solution without dye to obtain a mother solution of sunset yellow standard. Then, the mother solution of sunset yellow standard is further diluted with PBST without dye to multiple concentrations such as 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the original concentration, and PBST without dye is used as a blank control. Take 100 μL of each concentration solution and measure the absorbance of sunset yellow standard solution of different concentrations and blank PBST solution at a wavelength of 482 nm using an ELISA instrument. Calculate and draw a standard curve based on the absorbance and the known mother solution dye concentration.

[0050] Table 1

[0051]

[0052] According to the absorbance and corresponding dye concentration data in Table 1, the standard curve is obtained as follows: Figure 2 As shown:

[0053] (8) Based on the absorbance measured in step (6) and the standard curve calculated in step (7), the average food intake of each fruit fly on the two culture media A and B is calculated.

[0054] The calculation formula is:

[0055] Feed intake A = [(0.1028 × absorbance A × 2) / C × 1000] / n (μL)

[0056] Feed intake B = [(0.1028 × absorbance B × 2) / C × 1000] / n (μL)

[0057] Note: The absorbance A and absorbance B in the formula are the absorbance of the excrement solution after subtracting the blank. In this example, the absorbance A is: 0.232, and the absorbance B is: 0.059. In the formula, C is the concentration of the dye in the culture medium, which is: 1.5 mg / mL in this example. n is the number of fruit flies in each excrement measurement tube, which is: 10 / tube in this example. Substituting into the formula, the feed intake A = 3.180 μL, and the feed intake B = 0.809 μL are calculated.

[0058] (9) Based on the amount of food intake of the fruit flies for foods A and B calculated in step (8), calculate the preference index of the fruit flies for the two foods.

[0059] The calculation formula of the fruit fly's preference index for food A is:

[0060] Preference index A = (feed intake A - feed intake B) / (feed intake A + feed intake B)

[0061] The calculation formula of the fruit fly's preference index for food B is:

[0062] Preference index B = (feed intake B-feed intake A) / (feed intake A+feed intake B)

[0063] In this example, the calculated preference index A = (3.180-0.809) / (3.180+0.809) = 0.594

[0064] The calculated preference index B = (0.809–3.18) / (3.180+0.809) = -0.594

[0065] Test Example 1

[0066] It is known that fruit flies have feeding preferences for sugars, amino acids, and vitamins in food. Therefore, in order to verify the feasibility of this method to determine the feeding preferences of fruit flies, we used this method to determine the preferences of fruit flies for these substances in pure chemical culture medium. The results showed that under binary selective feeding conditions, fruit flies' food intake on culture medium without amino acids, sucrose, or vitamins was significantly lower than that on pure chemical culture medium, showing a significant feeding preference for these nutrients (such as Figure 4 This indicates that this method is suitable for detecting the feeding preference of fruit flies caused by a variety of nutritional components.

[0067] Test Example 2

[0068] It is known that under binary selective feeding conditions, fruit flies prefer to eat sucrose medium with relatively high concentrations rather than sucrose medium with lower concentrations. Therefore, in order to further test the sensitivity of this method in the determination of feeding preference, we measured the feeding preference of fruit flies for sucrose medium with different concentrations. The results showed that this method can not only detect the feeding preference of fruit flies under binary selective feeding conditions of two sucrose media with large concentration differences (for example: 50mM vs 20mM), but also can detect the feeding preference of fruit flies under binary selective feeding conditions of two sucrose media with very small concentration differences (for example: 50mM vs 45mM), which shows that this method has high sensitivity (such as Figure 5 shown).

[0069] The above embodiments are only preferred implementations of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for determining the feeding preference of fruit flies based on single-color marking of sunset yellow dye, comprising: (1) Two sets of feeding preference test tubes were used to separately label the two foods with 0.15% sunset yellow dye; (2) The fruit flies were kept in a feeding preference test tube for 24 hours, and then the sunset yellow dye in the excrement of the fruit flies after feeding was collected and dissolved in a buffer solution. The absorbance of the solution was measured to calculate the feeding preference of the fruit flies for the two foods.

2. The method according to claim 1, wherein: The step (1) of separately marking the two foods comprises: Prepare two culture media, A and B, add sunset yellow dye to half of each culture medium, and then distribute them into food cups, wherein food A without dye is paired with food B containing sunset yellow, and food B without dye is paired with food A containing sunset yellow.

3. The method according to claim 1, wherein: The buffer solution in step (2) is 2 mL of PBST solution.

4. The method according to claim 1, wherein: The calculation in step (2) of the feeding preference of fruit flies for the two foods includes: (2.1) Take 100 μL of each excrement dye solution, use a PBST solution without dye as a blank control, measure the absorbance of the solution at a wavelength of 482 nm using an ELISA reader, and subtract the absorbance of the blank control to obtain the absorbance A and absorbance B of the two excrement dye solutions A and B; (2.2) Weigh 10 mg of sunset yellow dye and dissolve it in another 10 ml of dye-free PBST solution to obtain a sunset yellow standard stock solution. Then, the sunset yellow standard stock solution is further diluted with dye-free PBST to multiple concentrations such as 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the original concentration, and the dye-free PBST is used as a blank control. 100 μL of each concentration solution is taken and the absorbance of the sunset yellow standard solution of different concentrations and the blank PBST solution at a wavelength of 482 nm is measured by an ELISA instrument. According to the absorbance and the known dye concentration of the mother solution, a standard curve is calculated and drawn; (2.3) Based on the measured absorbance and the calculated standard curve, calculate the average food intake of each fruit fly on culture media A and B; (2.4) Based on the calculated amount of food intake by fruit flies for foods A and B, calculate the preference index of fruit flies for the two foods.

5. The method according to claim 4, wherein: The calculation formula in step (2.3) is: Feed intake A = [(0.1028 × absorbance A × 2) / C × 1000] / n (μL); Feed intake B = [(0.1028 × absorbance B × 2) / C × 1000] / n (μL).

6. The method according to claim 4, wherein: The calculation formula of the preference index of fruit flies for food A in step (2.4) is: Preference index A = (feed intake A - feed intake B) / (feed intake A + feed intake B); The calculation formula of the fruit fly's preference index for food B is: Preference index B = (feed intake B - feed intake A) / (feed intake A + feed intake B).

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