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Quantifying Drosophila food intake: comparative analysis of current methodology.

Deshpande SA, Carvalho GB, Amador A, Phillips AM, Hoxha S, Lizotte KJ, Ja WW - Nat. Methods (2014)

Bottom Line: Despite the prevalent use of Drosophila in laboratory research, precise measurements of food intake remain challenging in this model organism.We show that the CAFE and radioisotope labeling provide the most consistent results, have the highest sensitivity and can resolve differences in feeding that dye labeling and PE fail to distinguish.Understanding the strengths and limitations of methods for measuring food intake will greatly advance Drosophila studies of nutrition, behavior and disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Metabolism and Aging, The Scripps Research Institute, Jupiter, Florida, USA. [2].

ABSTRACT
Food intake is a fundamental parameter in animal studies. Despite the prevalent use of Drosophila in laboratory research, precise measurements of food intake remain challenging in this model organism. Here, we compare several common Drosophila feeding assays: the capillary feeder (CAFE), food labeling with a radioactive tracer or colorimetric dye and observations of proboscis extension (PE). We show that the CAFE and radioisotope labeling provide the most consistent results, have the highest sensitivity and can resolve differences in feeding that dye labeling and PE fail to distinguish. We conclude that performing the radiolabeling and CAFE assays in parallel is currently the best approach for quantifying Drosophila food intake. Understanding the strengths and limitations of methods for measuring food intake will greatly advance Drosophila studies of nutrition, behavior and disease.

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Feeding measurements using pairs of assays simultaneously. (a) Radio- and dye-labeling. Flies were fed solid food labeled with both 32P and dye and both labels were quantified after 15 min of feeding. (b) Radiolabeling and CAFE. Flies were fed 32P-labeled liquid diets presented in capillaries in the CAFE enclosure. Both direct liquid consumption and radioisotope accumulation were measured after 24 h. “Internal,” radioisotope measured in the animal bodies; “Excreted,” radioisotope measured on vial walls. Statistical comparisons were made using total consumption (Excreted + Internal). (c) Radiolabeling and PE. Flies were fed 32P-labeled solid food. Observations of active feeding were made for 1 h, after which flies were sacrificed and 32P accumulation was measured. For all data (means ± s.e.m.; n = number of vials, shown in white over black bars), Dahomey males were fed the indicated diets (1× = 2.5% yeast extract + 2.5% sucrose). For P > 0.05 (unpaired, two-tailed t-test), exact p values are shown; otherwise, **, P < 0.01; ***, P < 0.001.
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Figure 3: Feeding measurements using pairs of assays simultaneously. (a) Radio- and dye-labeling. Flies were fed solid food labeled with both 32P and dye and both labels were quantified after 15 min of feeding. (b) Radiolabeling and CAFE. Flies were fed 32P-labeled liquid diets presented in capillaries in the CAFE enclosure. Both direct liquid consumption and radioisotope accumulation were measured after 24 h. “Internal,” radioisotope measured in the animal bodies; “Excreted,” radioisotope measured on vial walls. Statistical comparisons were made using total consumption (Excreted + Internal). (c) Radiolabeling and PE. Flies were fed 32P-labeled solid food. Observations of active feeding were made for 1 h, after which flies were sacrificed and 32P accumulation was measured. For all data (means ± s.e.m.; n = number of vials, shown in white over black bars), Dahomey males were fed the indicated diets (1× = 2.5% yeast extract + 2.5% sucrose). For P > 0.05 (unpaired, two-tailed t-test), exact p values are shown; otherwise, **, P < 0.01; ***, P < 0.001.

Mentions: We fed flies food labeled with both dye and a radiolabel. Intake values were similar in both assays, but only radiolabeling was able to distinguish between 1× and 2× foods (Fig. 3a). As discussed above, substantial biological variability is expected in brief assay periods. Even with extremely sensitive labels such as radioisotopes, intake should ideally not be inferred from very short-term assays. Long-term assays should systematically be given preference.


Quantifying Drosophila food intake: comparative analysis of current methodology.

Deshpande SA, Carvalho GB, Amador A, Phillips AM, Hoxha S, Lizotte KJ, Ja WW - Nat. Methods (2014)

Feeding measurements using pairs of assays simultaneously. (a) Radio- and dye-labeling. Flies were fed solid food labeled with both 32P and dye and both labels were quantified after 15 min of feeding. (b) Radiolabeling and CAFE. Flies were fed 32P-labeled liquid diets presented in capillaries in the CAFE enclosure. Both direct liquid consumption and radioisotope accumulation were measured after 24 h. “Internal,” radioisotope measured in the animal bodies; “Excreted,” radioisotope measured on vial walls. Statistical comparisons were made using total consumption (Excreted + Internal). (c) Radiolabeling and PE. Flies were fed 32P-labeled solid food. Observations of active feeding were made for 1 h, after which flies were sacrificed and 32P accumulation was measured. For all data (means ± s.e.m.; n = number of vials, shown in white over black bars), Dahomey males were fed the indicated diets (1× = 2.5% yeast extract + 2.5% sucrose). For P > 0.05 (unpaired, two-tailed t-test), exact p values are shown; otherwise, **, P < 0.01; ***, P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: Feeding measurements using pairs of assays simultaneously. (a) Radio- and dye-labeling. Flies were fed solid food labeled with both 32P and dye and both labels were quantified after 15 min of feeding. (b) Radiolabeling and CAFE. Flies were fed 32P-labeled liquid diets presented in capillaries in the CAFE enclosure. Both direct liquid consumption and radioisotope accumulation were measured after 24 h. “Internal,” radioisotope measured in the animal bodies; “Excreted,” radioisotope measured on vial walls. Statistical comparisons were made using total consumption (Excreted + Internal). (c) Radiolabeling and PE. Flies were fed 32P-labeled solid food. Observations of active feeding were made for 1 h, after which flies were sacrificed and 32P accumulation was measured. For all data (means ± s.e.m.; n = number of vials, shown in white over black bars), Dahomey males were fed the indicated diets (1× = 2.5% yeast extract + 2.5% sucrose). For P > 0.05 (unpaired, two-tailed t-test), exact p values are shown; otherwise, **, P < 0.01; ***, P < 0.001.
Mentions: We fed flies food labeled with both dye and a radiolabel. Intake values were similar in both assays, but only radiolabeling was able to distinguish between 1× and 2× foods (Fig. 3a). As discussed above, substantial biological variability is expected in brief assay periods. Even with extremely sensitive labels such as radioisotopes, intake should ideally not be inferred from very short-term assays. Long-term assays should systematically be given preference.

Bottom Line: Despite the prevalent use of Drosophila in laboratory research, precise measurements of food intake remain challenging in this model organism.We show that the CAFE and radioisotope labeling provide the most consistent results, have the highest sensitivity and can resolve differences in feeding that dye labeling and PE fail to distinguish.Understanding the strengths and limitations of methods for measuring food intake will greatly advance Drosophila studies of nutrition, behavior and disease.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Metabolism and Aging, The Scripps Research Institute, Jupiter, Florida, USA. [2].

ABSTRACT
Food intake is a fundamental parameter in animal studies. Despite the prevalent use of Drosophila in laboratory research, precise measurements of food intake remain challenging in this model organism. Here, we compare several common Drosophila feeding assays: the capillary feeder (CAFE), food labeling with a radioactive tracer or colorimetric dye and observations of proboscis extension (PE). We show that the CAFE and radioisotope labeling provide the most consistent results, have the highest sensitivity and can resolve differences in feeding that dye labeling and PE fail to distinguish. We conclude that performing the radiolabeling and CAFE assays in parallel is currently the best approach for quantifying Drosophila food intake. Understanding the strengths and limitations of methods for measuring food intake will greatly advance Drosophila studies of nutrition, behavior and disease.

Show MeSH