Method for simultaneously detecting food consumption and excrement area of drosophila melanogaster

Abstract

The invention discloses a method for simultaneously detecting food consumption and excrement area of fruit flies, which adopts a self-made fruit fly excrement collection box to simultaneously detect the food consumption and excrement area of the fruit flies, the fruit flies are fed in the excrement collection box, and the main body part of the excrement collection box is an arc-shaped excrement collection tray. And a food cup with the inner diameter of 6mm is mounted in the center of the excrement collecting tray. The food cup is filled with solid food containing 0.2% of brilliant blue dye. After the drosophila melanogaster eats food containing dye, as the brilliant blue dye cannot be absorbed by the drosophila melanogaster, the dye ingested by the drosophila melanogaster can be completely excreted into the arc-shaped excrement collecting tray. The total area of the drosophila melanogaster excrement can be calculated by taking a picture of the excrement collecting tray after the drosophila melanogaster eats. Then, the dye in the excrement on the excrement collecting tray is re-dissolved by using a PBST solution, the absorbance is measured, and the food consumption of the drosophila melanogaster can be calculated. Through the steps, the food consumption and the excrement area of the drosophila melanogaster can be measured at the same time.

Description

Technical Field

[0001] The invention belongs to the field of fruit fly food intake and excrement detection methods, and in particular relates to a method for simultaneously detecting fruit fly food intake and excrement area. Background Art

[0002] Drosophila is a commonly used model animal in genetics, nutritional physiology and behavioral research. Due to the high conservation of the regulatory mechanism of intestinal nutritional metabolism in Drosophila and humans, Drosophila has been widely used in recent years for research related to intestinal nutritional metabolism and development. Diarrhea is an important manifestation of imbalance in intestinal metabolic homeostasis, and its severity can be used as one of the physiological indicators reflecting intestinal functional damage. Diarrhea in Drosophila is specifically manifested as an increase in the area of ​​excrement. Given that the amount of Drosophila excrement is directly related to the amount of food intake, when evaluating the degree of Drosophila diarrhea, the surface area of ​​excrement and the amount of food intake must also be measured simultaneously to accurately and quantitatively reflect the specific degree of diarrhea. However, there is currently no method that can achieve this goal at the same time. To solve this problem, we developed a method to simultaneously detect the amount of food intake and excrement area of ​​Drosophila. Summary of the invention

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

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

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

[0006] (1) The fruit flies are raised in an excrement collection box, the main part of which 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) Fruit flies are raised in an excrement collection box equipped with a food cup, and their excrement is collected.

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

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

[0010] Furthermore, in step (1), 5 fruit flies are placed in the excrement collection plate; and 0.25 ml of homemade culture medium is contained in the food cup.

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

[0012] Prepare a test culture medium containing 0.7% agar. When the culture medium cools to 60°C, add 2 mg of 0.2% brilliant blue dye to every 1 ml of the culture medium. Mix well and dispense into food cups. Wait for the culture medium to cool and solidify before use.

[0013] Furthermore, in step (2), the feeding is to allow the fruit flies to eat freely; and the feeding time is 24 hours.

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

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

[0016] Furthermore, the diarrhea index in step (4) = excrement area ÷ food intake.

[0017] The beneficial effects of the present invention are:

[0018] The specific excrement collection box developed by the present invention can simultaneously measure the food intake and excrement of fruit flies at multiple time points without killing the test fruit flies, so it is suitable for studying the long-term effects of drugs, nutrition and other intervention methods on the appetite and diarrhea degree of fruit flies. At the same time, the device is low in cost, and the core component, the excrement collection tray, can be mass-produced by 3D printing technology. Therefore, the present invention has great application prospects in biological research. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] (a) The excrement collection box is composed of seven specific parts, including: four 3mm nuts (1), a transparent acrylic plate (2), a rubber sealing ring (3), an excrement collection plate (4), four 3mm screws (5), a food cup (6), and a rubber stopper (7);

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

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

[0023] (d) is a top view of the assembled excrement collection box;

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

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

[0026] Figure 2 The figure is 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 57 mm long and wide, and 20 mm high. A circular step (1) with a diameter of 8 mm and a height of 0.5 mm is designed at each of the four corners of the front side. In the middle of the step is a circular hole (2) with a diameter of 3 mm. In the center of the excrement collection tray is an arc-shaped circular disk (5) with a diameter of 40 mm. In the center of the circular disk is a circular hole (3) with a diameter of 7 mm. The edge of the circular disk is a circular groove (4) with an outer diameter of 50 mm, an inner diameter of 44 mm, a width of 3 mm, and a depth of 1.5 mm.

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

[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 acrylic plate, food cup, nut, screw, rubber seal and rubber plug, where:

[0033] (ab) The transparent acrylic plate has a length and width of 57 mm, a thickness of 2 mm, and a circular hole (1) with a diameter of 3 mm at each corner.

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

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

[0036] (e) The screw length is 14mm, the diameter is 3mm, the top cap diameter is 6mm, and the thickness is 2mm.

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

[0038] Figure 4 This is a picture of the excrement collection box and how to use it, including:

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

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

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

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

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

[0044] (6) Remove the funnel on 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 facing upwards in an incubator for 24 hours;

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

[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 into the excrement collection tray (14).

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

[0051] (16) Shake the fecal collection box to dissolve the dye in PBST. Calculate the food 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 feeding amount and excrement area; where:

[0053] (a) The changes in the area and morphology of Drosophila excrement from the first day to the ninth day under normal culture medium and cholesterol-deficient culture medium conditions;

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

[0055] (c) is a graph showing the excrement area of ​​Drosophila under normal medium and cholesterol-deficient medium conditions;

[0056] (d) Diarrhea index of fruit flies raised in normal medium and cholesterol-deficient medium.

[0057] Figure 6 To take photos of the excrement in the excrement collection tray and handle the steps, including:

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

[0059] (b) is to convert the photo into a grayscale image, in which case the excrement appears gray-black;

[0060] (c) Using ImageJ image processing software to identify the gray-black area and convert it to red, the number of pixels of excrement in the red area was measured;

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

[0062] Figure 7 This is a bright blue standard curve. DETAILED DESCRIPTION

[0063] 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.

[0064] Example

[0065] (1) Assembly of the excrement collection box: Assemble all the parts of the excrement collection box except the food cup (nuts, acrylic plate, excrement collection tray, screws) according to Figure 1 Assembly is complete. Installation steps: (1) Place the transparent acrylic plate on the excrement collection tray so that the four small holes on the four corners of the transparent acrylic plate are aligned with the four small holes on the four corners of the excrement collection tray. (2) Insert the four screws through the four small holes on the back of the excrement collection tray and through the transparent acrylic plate. (3) Screw the screws into the nuts to secure the transparent acrylic plate 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) to the medium, mix well and dispense into food cups, adding 0.25 ml of medium to each food cup. The medium in the food cup can be used after it cools and solidifies.

[0067] (3) Transfer the fruit flies into the excrement collection boxes through the circular hole in the center of the bottom of the excrement collection box, with 5 fruit flies placed in each excrement collection box.

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

[0069] (5) Place the excrement collection box with the front side facing up in an incubator and allow the fruit flies to eat freely for 24 hours.

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

[0071] (7) Take a photo of the blue excrement on the excrement collection plate and calculate the number of excrement pixels N on the excrement collection plate using ImageJ image processing software. 1 And the total number of pixels N of the entire excrement collection tray 2 (like Figure 6 As shown). It is known that the total area of ​​the excrement collection tray is 1781mm 2 , then the average excrement area of ​​each fruit fly is A=1781×(N 1 / N 2 ) / n. Where n is the number of fruit flies raised in the excrement collection box.

[0072] In this example, the number of fecal pixels N is measured. 1 = 18834 pixels, the number of pixels in the entire excrement collection tray N 2 = 960000 pixels, the number of fruit flies in the excrement collection box n = 5. Then the average excrement area of ​​each fruit fly is calculated to be A = 1781 × (18834 / 960000) / 5 = 6.988 mm 2 .

[0073] (8) Insert a rubber stopper of the same size as the food cup into the circular 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 on the excrement collection box, and further remove the transparent acrylic plate.

[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 (phosphate buffered saline containing 0.1% Tween-20) to the feces collection dish.

[0077] (12) Install the transparent acrylic plate back onto the excrement collection tray and tighten the nut to ensure that the transparent acrylic plate fits tightly against the rubber sealing ring.

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

[0079] (14) Remove the nut on the excrement collection box, and further remove the transparent acrylic plate.

[0080] (15) The absorbance of the PBST solution containing brilliant blue obtained in step (13) at 630 nm was measured using an ELISA instrument, and a PBST solution without dye was used as a blank control. In this example, the absorbance measured was 0.301 (blank subtracted).

[0081] (16) Weigh 10 mg of brilliant blue dye and dissolve it in another 10 ml of PBST solution without dye to obtain a brilliant blue standard stock solution. Then, the brilliant blue standard stock solution 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. Using PBST without dye as a blank control, the absorbance of brilliant blue standard solutions of different concentrations and blank PBST solutions at 630 nm is measured by an ELISA instrument. Based on the absorbance and the known dye concentration of the mother solution, a standard curve is calculated and drawn, such as Figure 7 shown.

[0082] Table 1. Absorbance of Brilliant Blue Standard Solutions at Different Concentrations

[0083]

[0084] (17) Calculate the amount of food consumed by the fruit fly 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. Where C is the concentration of the 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 to be [(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 food intake measured in step (17) and the excrement area measured in step (7). Diarrhea index = excrement area / food intake.

[0089] In this example, 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, which in turn causes diarrhea. Therefore, in order to verify the feasibility of using the excrement collection box to measure the food intake and excrement area of ​​fruit flies, we used this device to measure the food intake, excrement area and morphology of fruit flies raised in normal culture medium and cholesterol-deficient culture medium. The results are shown in Figure 2. Figure 5 The results showed that although there was no significant difference in the food intake of fruit flies under the two feeding 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 shape and area of ​​fruit fly excrement did not change significantly from the first day to the ninth day, but under cholesterol-deficient culture medium conditions, the area of ​​fruit fly excrement gradually increased and the shape became more irregular, reflecting the increase in the degree of diarrhea ( Figure 5 a, Figure 5 c). In line with this, the calculated diarrhea index also increased significantly ( Figure 5 d), thus demonstrating the feasibility of using this method to simultaneously measure the food intake and excrement area of ​​fruit flies.

[0092] 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 simultaneously detecting the amount of food consumed and the area of ​​excrement of fruit flies, comprising: (1) The fruit flies are raised in an excrement collection box, the main part of which is an arc-shaped excrement collection tray, and a food cup with an inner diameter of 6 mm is installed in the center of the excrement collection tray; (2) raising fruit flies in an excrement collection box equipped with a food cup and collecting excrement; (3) The blue excrement of the fruit fly 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 food intake of the fruit fly; (4) The diarrhea index of fruit flies is calculated based on the food intake and excrement area.

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

3. The method according to claim 2, wherein: The self-made culture medium is prepared by the following method: Prepare a test culture medium containing 0.7% agar. When the culture medium cools to 60°C, add 2 mg of 0.2% brilliant blue dye to every 1 ml of the culture medium. Mix well and dispense into food cups. Wait for the culture medium to cool and solidify before use.

4. The method according to claim 1, wherein: In step (2), the feeding is that the fruit flies are allowed to eat freely; 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 calculation 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 solution containing 0.1% Tween-20.

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

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