A method for simultaneously detecting fruit fly feeding and excretion area

Abstract

The application discloses a method for simultaneously detecting the food intake and excrement area of fruit flies, and adopts a self-made fruit fly excrement collecting box to simultaneously detect the food intake and excrement area of fruit flies. The fruit flies are bred in the excrement collecting box. The main body part of the excrement collecting box is an arc-shaped excrement collecting disc. A food cup with an inner diameter of 6 mm is arranged in the center of the excrement collecting disc. The food cup is filled with solid food containing 0.2% bright blue dye. When the fruit flies eat the food containing the dye, the bright blue dye cannot be absorbed by the fruit flies, so the dye ingested by the fruit flies is completely excreted into the arc-shaped excrement collecting disc. The total area of the excrement of the fruit flies can be calculated by taking a photo of the excrement collecting disc after the fruit flies eat the food. Then, the absorbance of the dye in the excrement on the excrement collecting disc is measured by redissolving the dye with a PBST solution, so that the food intake of the fruit flies can be calculated. Through the above steps, the food intake and the excrement area of the fruit flies can be simultaneously measured.

Description

Technical Field

[0001] This invention belongs to the field of methods for detecting fruit fly feed intake and excrement, specifically relating to a method for simultaneously detecting fruit fly feed intake and excrement area. Background Technology

[0002] Drosophila is a commonly used model organism in genetics, nutritional physiology, and behavioral research. Due to the high conservation of intestinal nutrient metabolism regulation mechanisms in both Drosophila and humans, Drosophila has been extensively used in recent years for research related to intestinal nutrient metabolism and development. Diarrhea is an important manifestation of intestinal metabolic homeostasis imbalance, and its severity can serve as one of the physiological indicators reflecting intestinal functional impairment. In Drosophila, diarrhea is specifically characterized by an increase in excrement surface area. Given that the amount of excrement in Drosophila is directly related to the amount of food consumed, it is necessary to simultaneously measure excrement surface area and food intake to accurately quantify the specific degree of diarrhea when assessing its severity. However, currently, there is no method that can simultaneously achieve this goal. To address this issue, we have developed a method for simultaneously detecting food intake and excrement surface area in Drosophila. Summary of the Invention

[0003] In view of the above shortcomings, the present invention provides a method for simultaneously detecting the amount of food consumed by fruit flies and the area of ​​their excrement, which is mainly based on a self-designed fruit fly excrement collection box.

[0004] This invention is specifically achieved through the following technical means:

[0005] This invention first discloses a method for simultaneously detecting the feed intake and excrement area of ​​fruit flies, comprising the following steps:

[0006] (1) Fruit flies are raised in an excrement collection box. The main body of the excrement collection box is an excrement collection tray, and a food cup with an inner diameter of 6 mm is installed in the center of the excrement collection tray.

[0007] (2) The fruit flies were raised in an excrement collection box equipped with a food cup, and their excrement was collected.

[0008] (3) The blue excrement of fruit flies was photographed and the total area of ​​the excrement was calculated. Then, the dye in the excrement was redissolved by adding PBST solution and the absorbance at 630 nm was measured to obtain the amount of food consumed by the fruit flies.

[0009] (4) The diarrhea index of fruit flies is calculated based on the amount of food consumed and the area of ​​excrement.

[0010] Further, in step (1), five fruit flies are placed in the excrement collection tray; the food cup contains 0.25 ml of homemade culture medium.

[0011] Furthermore, the self-made culture medium is prepared by the following method:

[0012] Prepare a test medium containing 0.7% agar. When the medium cools to 60°C, add 2 mg of 0.2% brilliant blue dye to each 1 ml of medium, mix well, and dispense into food cups. The medium is ready for use once it has cooled and solidified.

[0013] Furthermore, in step (2), the fruit flies are allowed free access to food; the feeding time is 24 hours.

[0014] Furthermore, the total area of ​​the excrement calculated in step (3) is obtained by using ImageJ image processing software.

[0015] Further, the amount of PBST solution added in step (3) is 2 mL; the PBST solution is a phosphate buffer containing 0.1% Tween-20.

[0016] Further, the diarrhea index in step (4) is calculated as: excrement area ÷ feed intake.

[0017] The beneficial effects of this invention are as follows:

[0018] The specific excrement collection box developed in this invention can simultaneously measure the feed intake and excrement of fruit flies at multiple time points without euthanizing them, making it suitable for studying the long-term effects of drugs, nutrition, and other interventions on the appetite and diarrhea severity of fruit flies. Furthermore, the device is inexpensive, and the core component, the excrement collection tray, can be mass-produced using 3D printing technology. Therefore, this invention has significant application prospects in biological research. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the structure and assembly of the excrement collection box of the present invention, wherein:

[0020] (a) The excrement collection box consists of 7 types of components, including: four 3mm nuts (1), one transparent acrylic sheet (2), one rubber sealing ring (3), one excrement collection tray (4), four 3mm screws (5), one food cup (6), and one rubber stopper (7).

[0021] (b) A schematic diagram of the assembled excrement collection box (excluding the rubber sealing ring and rubber stopper);

[0022] (c) A perspective view of the assembled excrement collection box;

[0023] (d) Top view of the assembled excrement collection box;

[0024] (e) A bottom view of the assembled excrement collection box;

[0025] (f) is a side view of the assembled excrement collection box.

[0026] Figure 2 A schematic diagram of the specific structure of the excrement collection tray (4), wherein:

[0027] (a) is a top view of the excrement collection tray. The excrement collection tray is 57mm long and wide and 20mm high. There is a circular step with a diameter of 8mm and a height of 0.5mm at each of the four corners of the front (1). There is a circular hole with a diameter of 3mm in the middle of the step (2). The center of the excrement collection tray is an arc-shaped disc with a diameter of 40mm (5). There is a circular hole with a diameter of 7mm in the center of the disc (3). The edge of the disc is a circular groove with an outer diameter of 50mm, an inner diameter of 44mm, a width of 3mm and a depth of 1.5mm (4).

[0028] (b) is a bottom view of the excrement collection box. The bottom center of the excrement collection tray has an arc-shaped protrusion (7) with a diameter of 40 mm, and a sleeve (6) with an outer diameter of 14 mm, an inner diameter of 7 mm, and a height of 6 mm around the central circular hole (3). The square frame (8) of the excrement collection tray has a thickness of 2.5 mm.

[0029] (c) is a side view of the excrement collection tray.

[0030] (d) is a front perspective view of the excrement collection tray.

[0031] (e) is a perspective view of the back of the excrement collection tray.

[0032] Figure 3 This is a schematic diagram of the structure of an acrylic sheet, a food cup, a nut, a screw, a rubber sealing ring, and a rubber stopper, where:

[0033] (ab) The transparent acrylic sheet is 57mm long and wide, 2mm thick, and has a 3mm diameter hole at each of the four corners (1).

[0034] (c) The outer diameter of the food cup is 7mm, the inner diameter is 6mm, the height of the cup wall (2) is 10mm, the thickness of the cup wall is 0.5mm, and the bottom is a base (3) with a diameter of 9mm and a thickness of 0.5mm.

[0035] (d) The nut has an outer diameter of 10mm, an inner diameter of 3mm, a thickness of 8mm, and an internal thread hole depth of 6mm.

[0036] (e) The screw is 14mm long, 3mm in diameter, and has a cap with a diameter of 6mm and a thickness of 2mm.

[0037] (fg) The outer diameter of the rubber sealing ring is 50mm, the inner diameter is 44mm, and the thickness is 3mm. (h) The outer diameter of the rubber stopper is 7mm, the inner diameter is 4mm, the stopper height is 10mm, the stopper wall thickness is 1.5mm, and the bottom is a base with a diameter of 11mm and a thickness of 2mm.

[0038] Figure 4 The image shows the actual product of the excrement collection box and its usage instructions, including:

[0039] (1) A front view of the excrement collection tray;

[0040] (2) A view of the back of the excrement collection tray;

[0041] (3) Insert the screws into the holes at the four corners of the excrement collection tray, and then install the transparent acrylic cover onto the excrement collection tray.

[0042] (4) Install and tighten the nuts;

[0043] (5) Invert the excrement collection box and use a funnel to transfer the fruit flies into the excrement collection box;

[0044] (6) Remove the funnel from the excrement collection box and immediately insert the food cup into the hole on the back of the excrement collection tray;

[0045] (7) Place the excrement collection box upside down in the incubator for 24 hours;

[0046] (8, 9) Remove the food cup, move the fruit flies out of the excrement collection box, take a picture of the excrement containing dye on the excrement collection tray, and calculate the area of ​​the excrement.

[0047] (10 and 11) Insert the rubber stopper (10) into the hole at the bottom of the excrement collection tray (11).

[0048] (12) Remove the nuts and transparent acrylic cover.

[0049] (13, 14) Place a rubber sealing ring (13) in the annular groove, and then add 2 mL of PBST to the excrement collection tray (14).

[0050] (15) Place the acrylic cover back onto the excrement collection tray and tighten the nut.

[0051] (16) Shake the excrement collection box to dissolve the dye in PBST. Calculate the feed intake by measuring the absorbance of the solution at 630 nm.

[0052] Figure 5 This is an example of using an excrement collection box to simultaneously measure fruit fly feed intake and excrement area; where:

[0053] (a) A graph showing the changes in the area and morphology of excrement from day one to day nine of fruit flies under normal culture medium and cholesterol-deficient culture medium conditions.

[0054] (b) is a graph showing the feed intake of fruit flies under normal culture medium and cholesterol-deficient culture medium conditions;

[0055] (c) is a diagram showing the area of ​​excrement in fruit flies under normal culture medium and cholesterol-deficient culture medium conditions;

[0056] (d) is a graph showing the diarrhea index of fruit flies under normal culture medium and cholesterol-deficient culture medium conditions.

[0057] Figure 6 The steps for photographing and processing excrement in the excrement collection tray include:

[0058] (a) is a waste collection tray containing blue excrement;

[0059] (b) To convert the photo to grayscale, the excrement appears as gray-black;

[0060] (c) To use ImageJ image processing software to identify gray-black areas and convert them to red, the number of pixels of excrement in the red area was measured.

[0061] (d) To further measure the number of pixels in the entire excrement collection tray.

[0062] Figure 7 This is a standard curve graph for bright blue. Detailed Implementation

[0063] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. However, it should be understood that the embodiments and drawings are for illustrative purposes only and do not constitute any limitation on the scope of protection of the present invention. All reasonable modifications and combinations included within the scope of the inventive spirit of the present invention fall within the scope of protection of the present invention.

[0064] Example

[0065] (1) Assembly of the excrement collection box: Assemble all parts of the excrement collection box except for the food cup (nuts, acrylic sheet, excrement collection tray, screws) according to... Figure 1 Assembly complete. Installation steps: (1) Place the transparent acrylic sheet on the excrement collection tray, aligning the four small holes at the four corners of the transparent acrylic sheet with the four small holes at the four corners of the excrement collection tray. (2) Insert the four screws through the four small holes at the four corners of the back of the excrement collection tray, passing them through the transparent acrylic sheet. (3) Tighten the screws into the nuts to secure the transparent acrylic sheet to the excrement collection tray.

[0066] (2) Prepare a test medium containing 0.7% agar. When the medium cools to 60 degrees Celsius, add 0.2% brilliant blue dye (2 mg per 1 ml of medium), mix well, and dispense into food cups, adding 0.25 ml of medium to each food cup. The medium can be used after it cools and solidifies in the food cups.

[0067] (3) Transfer the fruit flies into the excrement collection box through the round hole in the center of the bottom of the excrement collection box, and put 5 fruit flies into each excrement collection box.

[0068] (4) Insert the food cup containing the culture medium into the round hole in the center of the bottom of the excrement collection box and install it into the excrement collection box.

[0069] (5) Place the excrement collection box face up in the incubator and allow the fruit flies to feed freely for 24 hours.

[0070] (6) After 24 hours of feeding, remove the food cup from the excrement collection box and transfer the fruit flies out of the excrement collection box.

[0071] (7) Take a picture of the blue excrement on the excrement collection tray, and use ImageJ image processing software to calculate the number of pixels N1 of the excrement on the collection tray and the total number of pixels N2 of the entire collection tray (e.g., ...). Figure 6 (As shown). The total area of ​​the excrement collection tray is known to be 1781 mm². 2 Therefore, the average excrement area per fruit fly is A = 1781 × (N1 / N2) / n. Where n is the number of fruit flies kept in the excrement collection box.

[0072] In this example, the number of excrement pixels was measured to be N1 = 18834 pixels, the total number of pixels in the excrement collection tray was N2 = 960000 pixels, and the number of fruit flies in the excrement collection box was n = 5. Therefore, the average excrement area per fruit fly was calculated to be A = 1781 × (18834 / 960000) / 5 = 6.988 mm². 2 .

[0073] (8) Insert a rubber stopper the same size as the food cup into the round hole in the center of the bottom of the excrement collection box to seal the bottom of the excrement collection tray.

[0074] (9) Remove the nut from the excrement collection box and then remove the transparent acrylic sheet.

[0075] (10) Place a rubber sealing ring into the annular groove on the excrement collection tray.

[0076] (11) Add 2 ml of 0.1% PBST solution (containing 0.1% Tween-20 phosphate buffer) to the excrement collection plate.

[0077] (12) Install the transparent acrylic sheet back onto the excrement collection tray and tighten the nut so that the transparent acrylic sheet is tightly attached to the rubber sealing ring.

[0078] (13) Shake the excrement collection box containing PBST solution to dissolve the brilliant blue dye in the excrement into the PBST.

[0079] (14) Remove the nut on the excrement collection box and then remove the transparent acrylic sheet.

[0080] (15) The absorbance of the PBST solution containing brilliant blue obtained in step (13) at 630 nm was measured using an ELISA reader, with a PBST solution without dye as a blank control. In this example, the absorbance was measured to be 0.301 (after subtracting the blank).

[0081] (16) Weigh 10 mg of Brilliant Blue dye and dissolve it in another 10 ml of dye-free PBST solution to obtain Brilliant Blue standard stock solution. Further dilute this Brilliant Blue standard stock solution with dye-free PBST to multiple concentrations (1 / 2, 1 / 4, 1 / 8, 1 / 16, etc.), using dye-free PBST as a blank control. Measure the absorbance at 630 nm of different concentrations of Brilliant Blue standard solution and blank PBST solution using an ELISA reader. Calculate and plot a standard curve based on this absorbance and the known dye concentration of the stock solution, as shown below. Figure 7 As shown.

[0082] Table 1. Absorbance of Brilliant Blue standard solutions at different concentrations

[0083]

[0084] (17) Calculate the feed intake of the fruit flies based on the standard curve obtained in step (16) and the absorbance of the excrement solution measured in step (15). The calculation formula is:

[0085] Feed intake = [(0.0337 × absorbance × 2) / C × 1000] / n (μL)

[0086] Where n is the number of fruit flies raised in the excrement collection box. In the formula, C is the concentration of dye in the culture medium.

[0087] In this example, n = 5, the absorbance of the excrement solution is 0.301, and the dye concentration C in the culture medium is 2 mg / mL. Therefore, the feed intake is calculated as [(0.0337 × 0.301 × 2) / 2 × 1000] / 5 (μL) = 2.029 μL.

[0088] (18) Calculate the diarrhea index of the fruit fly based on the amount of food consumed in step (17) and the area of ​​excrement measured in step (7). Diarrhea index = area of ​​excrement / amount of food consumed.

[0089] In this example, the diarrhea index = 6.988 mm. 2 / 2.029μL=3.444mm 2 / μL.

[0090] Application examples

[0091] It is known that cholesterol deficiency can lead to intestinal barrier damage, resulting in diarrhea. Therefore, to verify the feasibility of using an excrement collection box to measure feed intake and excrement area in fruit flies, we used this device to measure feed intake, excrement area, and morphology in fruit flies fed on normal and cholesterol-deficient culture media. The results are as follows: Figure 5 The results showed that, although there was no significant difference in feed intake between the two rearing conditions ( Figure 5 b), but there are significant differences in the shape and area of ​​excrement ( Figure 5 a, Figure 5 c). Under normal culture medium conditions, the morphology and area of ​​fruit fly excrement did not change significantly from day one to day nine. However, under cholesterol-deficient culture medium conditions, the area of ​​fruit fly excrement gradually increased, and the morphology became more irregular, reflecting an increase in the severity of diarrhea. Figure 5 a, Figure 5 c). Consistent with this, the calculated diarrhea index also increased significantly. Figure 5 d), thus proving the feasibility of using this method to simultaneously determine the feed intake and excrement area of ​​fruit flies.

[0092] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting the amount of food consumed and the area of ​​excrement in fruit flies, comprising: (1) Assembly of the excrement collection box: Place the transparent acrylic sheet on the excrement collection tray, so that the four small holes at the four corners of the transparent acrylic sheet are aligned with the four small holes at the four corners of the excrement collection tray. Insert four screws through the four small holes at the four corners of the back of the excrement collection tray, passing them through the transparent acrylic sheet. Tighten the screws into the nuts to secure the transparent acrylic sheet to the excrement collection tray. Transfer the fruit flies into the excrement collection box through the round hole in the center of the bottom. Insert the food cup containing culture medium into the excrement collection box through the round hole in the center of the bottom. The main body of the excrement collection box is an arc-shaped excrement collection tray with a food cup with an inner diameter of 6mm installed in the center. After 24 hours of feeding, remove the food cup from the excrement collection box and transfer the fruit flies out. Insert a rubber stopper of the same size as the food cup into the round hole in the center of the bottom of the excrement collection box to seal the bottom of the excrement collection tray. Remove the nuts on the excrement collection box and further remove the transparent acrylic sheet. Place a rubber sealing ring into the annular groove on the excrement collection tray. (2) The fruit flies were raised in an excrement collection box equipped with a food cup, and their excrement was collected. (3) The blue excrement of fruit flies was photographed and the total area of ​​the excrement was calculated. Then, the dye in the excrement was redissolved by adding PBST solution and the absorbance at 630 nm was measured to calculate the amount of food consumed by the fruit flies. (4) The diarrhea index of fruit flies is calculated based on the amount of food consumed and the area of ​​excrement.

2. The method according to claim 1, wherein: In step (1), five fruit flies are placed in the excrement collection tray. The food cup contained 0.25 ml of homemade culture medium.

3. The method according to claim 2, wherein: The self-made culture medium was prepared by the following method: Prepare a test medium containing 0.7% agar. When the medium cools to 60°C, add 2 mg of 0.2% brilliant blue dye to each 1 ml of medium, mix well, and dispense into food cups. The medium is ready for use once it has cooled and solidified.

4. The method according to claim 1, wherein: The feeding method described in step (1) involves allowing fruit flies free access to food; The feeding time is 24 hours.

5. The method according to claim 1, wherein: The total area of ​​the excrement calculated in step (3) is obtained by using ImageJ image processing software.

6. The method according to claim 1, wherein: The amount of PBST solution added in step (3) is 2 mL; The PBST solution is a phosphate buffer containing 0.1% Tween-20.

7. The method according to claim 1, wherein: The diarrhea index in step (4) is calculated as: excrement area ÷ food intake.

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