Gender-specific prandial response to dietary restriction and oxidative stress in Drosophila melanogaster

Abstract

Drosophila melanogaster is ideal for studying lifespan modulated by dietary restriction (DR) and oxidative stress, and also for screening prolongevity compounds. It is critical to measure food intake in the aforementioned studies. Current methods, however, overlook the amount of the food excreted out of the flies as feces or deposited in eggs. Here we describe a feeding method using a radioactive tracer to measure gender-specific food intake, retention and excretion in response to DR and oxidative stress to account for all the ingested food. Flies were fed a full, restricted or paraquat-containing diet. The radioactivity values of the food in fly bodies, feces and eggs were measured separately after a 24-hr feeding. Food intake was calculated as the sum of these measurements. We found that most of the tracer in the ingested food was retained in the fly bodies and < 8% of the tracer was excreted out of the flies as feces and eggs in the case of females during a 24-hr feeding. Under a DR condition, flies increased food intake in volume to compensate for the reduction of calorie content in the diet and also slightly increased excretion. Under an oxidative stress condition, flies reduced both food intake and excretion. Under all the tested dietary conditions, males ingested and excreted 3-5 fold less food than females. This study describes an accurate method to measure food intake and provides a basis to further investigate prandial response to DR and prolongevity interventions in invertebrates.

Figure 1

Measurement of food intake. (A) Distribution of the ingested food tracer. ³²P-dCTP was used as the tracer. (B) Lifespan of male and female flies fed the full (1x SY) or DR diet (0.25x SY). (C) Feeding setup. Food was provided to flies in a tube inserted into the plug of a tilted vial. 1.5% agar in the bottom of the vial was provided for females to lay eggs. (D) Percentage of eggs laid on the agar only surface. “f” represents female; “1x” refers to the full diet; “0.25x” refers to the DR diet.

Figure 2

Prandial response of flies to the full, DR and paraquat diets. (A) Food retention in a fly. (B) Tracer in excretion. (C) Tracer in eggs from a female. (D) Total food tracer release. Each measurement was repeated three times with three vials each housing approximately 20 flies. For the full diet represented by 1x, total number of males (N-male) was 60 for the three replicates and total number of females (N-female) was 57; for the DR diet (0.25x), N-male = 60; N-female = 58; for the paraquat diet, N-male = 59; N-female = 58; “f” represents female and “m” refers to male. “⁺p” represents addition of paraquat to the diet. The amount of food in µl shown on Y axis was calculated based on the amount of food tracer. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Total food intake. (A) Volume of ingested food. (B) Percentage of calorie intake for flies under DR and oxidative stress when compared to that under the full diet, which was normalized to 100% separately for males and females. The comparison was conducted within each sex. The abbreviations are the same as those in Figure 2. *p < 0.05; **p < 0.01.

Figure 4

Distribution of fecal spots and eggs. (A) Fecal spot counts of flies under the diet, DR and paraquat diets. (B) Percentage of total eggs laid on the agar only surface under three dietary conditions. The numbers of eggs laid by one female on the bottom agar only surface and the top food surface on average are 17.4 ± 2.2 and 0.6 ± 0.3 respectively for the full diet (1x), 20.8 ± 8.8 and 1.1 ± 0.4 respectively for the DR diet (0.25x), and 12.5 ± 0.6 and 1.4 ± 0.3 respectively for the paraquat diet (1 x +p). Each measurement was repeated three times with three vials each containing approximately 20 females. The abbreviations are the same as those in Figure 2.