Parallelized, real-time, metabolic-rate measurements from individual Drosophila

Abstract

Significant recent evidence suggests that metabolism is intricately linked to the regulation and dysfunction of complex cellular and physiological responses ranging from altered metabolic programs in cancers and aging to circadian rhythms and molecular clocks. While the metabolic pathways and their fundamental control mechanisms are well established, the precise cellular mechanisms underpinning, for example, enzymatic pathway control, substrate preferences or metabolic rates, remain far less certain. Comprehensive, continuous metabolic studies on model organisms, such as the fruit fly Drosophila melanogaster, may provide a critical tool for deciphering these complex physiological responses. Here, we describe the development of a high-resolution calorimeter, which combines sensitive thermometry with optical imaging to concurrently perform measurements of the metabolic rate of ten individual flies, in real-time, with ~100 nW resolution. Using this calorimeter we have measured the mass-specific metabolic rates of flies of different genotypes, ages, and flies fed with different diets. This powerful new approach enables systematic studies of the metabolic regulation related to cellular and physiological function and disease mechanisms.

Figure 1

Calorimetric platform. (a) Schematic illustration of the calorimeter’s working principle. The fly is contained at the center of a glass tube (2 × 2 mm² inner cross section). The high-sensitivity thermistor detects the small temperature increase, ΔT_sense, due to the fly’s heat output, q_metabolic. The thermal conductance of the glass tube, G_tube, is the primary pathway for heat transfer as shown in the thermal resistance network. (b) Rendering of the entire system. Two independently-controlled sets of Peltier modules maintain the temperature of the inner and outer thermal shields. Two CCD cameras optically image the ten measurement chambers. All ten flies breathe air from a temperature-controlled, humidified air reservoir (Methods). (c) Detailed view of selected calorimeter tubes with a sensing thermistor for measuring ΔT_sense and a heating thermistor for calibrating the conductance (G_tube). (Methods). Copper tape ensures isothermal conditions at the center of the tube. (d) Plot of the temperature rise (ΔT_sense) vs. controlled electrical heat input (q_Joule) for a representative calorimeter showing that heat flows as small as 100 nW can be reliably detected. Inset: Same as (d) but on linear axes. The inverse of the slope yields G_tube = 2.02 mW/K.

Figure 2

Metabolic measurements. (a) Time traces showing the speed of locomotion (upper panel) and heat production (lower panel) of a single Canton S (female, 3 days after eclosion) over the course of an experiment. The black dashed line in the upper panel delineates the 4 mm/min threshold for defining the rest state, and the green portions of the heat production trace (lower panel) indicate the times for which the rest condition is met. The dashed green line indicates the average basal heat production while the black dashed-dotted line represents the total average heat production for this fly. (b) Heat production plotted against activity level for the same single Canton S fly from (a). The dashed vertical line indicates the threshold for the rest condition. (c) Heat production data for a population of 50 individual Canton S flies averaged during periods of high (>50 mm/min) and low (<4 mm/min) activity. The horizontal line represents the mean for a sample. Box boundaries indicate the standard error of the mean, and error bars represent the standard deviation of the sample. The open circles to the left of the boxes signify the average heat production for the individual flies. ***p < 0.001. (d) Heat production data for ten flies (A–J) during a single experiment. Fly F is shown in (a,b).

Figure 3

Basal and average metabolic heat production rates. (a–c) Basal heat production as measured for: (a) the three Drosophila genotypes considered: Canton S, w¹¹¹⁸, and yw, (b) Canton S flies of different ages, and (c) Canton S flies on high calorie (HCD), normal (ND) and restricted diets (RD) at different ages. (d–f) Mass-specific basal metabolic rate of flies (see Supplementary Fig. S2) of different: (d) genotypes, (e) ages, and (f) dietary conditions. (g–i) Mass-specific average heat production for the same flies as in (a–c). Shown are the mean (horizontal line), standard error (box) and standard deviation (error bars). The open circles to the left of the boxes represent the average heat production for individual flies and N indicates the sample size. ANOVA results are indicated on each panel; Tukey’s tests are indicated pairwise: ^*p < 0.01, ^**p < 0.005, ^***p < 0.001.