Mated Drosophila melanogaster females consume more amino acids during the dark phase.

To maintain homeostasis, animals must ingest appropriate quantities, determined by their internal nutritional state, of suitable nutrients. In the fruit fly Drosophila melanogaster, an amino acid deficit induces a specific appetite for amino acids and thus results in their increased consumption. Although multiple processes of physiology, metabolism, and behavior are under circadian control in many organisms, it is unclear whether the circadian clock also modulates such motivated behavior driven by an internal need. Differences in levels of amino acid consumption by flies between the light and dark phases of the day:night cycle were examined using a capillary feeder assay following amino acid deprivation. Female flies exhibited increased consumption of amino acids during the dark phase compared with the light phase. Investigation of mutants lacking a functional period gene (per0), a well-characterized clock gene in Drosophila, found no difference between the light and dark phases in amino acid consumption by per0 flies. Furthermore, increased consumption of amino acids during the dark phase was observed in mated but not in virgin females, which strongly suggested that mating is involved in the rhythmic modulation of amino acid intake. Egg production, which is induced by mating, did not affect the rhythmic change in amino acid consumption, although egg-laying behavior showed a per0-dependent change in rhythm. Elevated consumption of amino acids during the dark phase was partly induced by the action of a seminal protein, sex peptide (SP), on the sex peptide receptor (SPR) in females. Moreover, we showed that the increased consumption of amino acids during the dark phase is induced in mated females independently of their internal level of amino acids. These results suggest that a post-mating SP/SPR signal elevates amino acid consumption during the dark phase via the circadian clock

Mated <i>Drosophila melanogaster</i> females consume more amino acids during the dark phase

<div><p>To maintain homeostasis, animals must ingest appropriate quantities, determined by their internal nutritional state, of suitable nutrients. In the fruit fly <i>Drosophila melanogaster</i>, an amino acid deficit induces a specific appetite for amino acids and thus results in their increased consumption. Although multiple processes of physiology, metabolism, and behavior are under circadian control in many organisms, it is unclear whether the circadian clock also modulates such motivated behavior driven by an internal need. Differences in levels of amino acid consumption by flies between the light and dark phases of the day:night cycle were examined using a capillary feeder assay following amino acid deprivation. Female flies exhibited increased consumption of amino acids during the dark phase compared with the light phase. Investigation of mutants lacking a functional <i>period</i> gene (<i>per</i>⁰), a well-characterized clock gene in <i>Drosophila</i>, found no difference between the light and dark phases in amino acid consumption by <i>per</i>⁰ flies. Furthermore, increased consumption of amino acids during the dark phase was observed in mated but not in virgin females, which strongly suggested that mating is involved in the rhythmic modulation of amino acid intake. Egg production, which is induced by mating, did not affect the rhythmic change in amino acid consumption, although egg-laying behavior showed a <i>per</i>⁰-dependent change in rhythm. Elevated consumption of amino acids during the dark phase was partly induced by the action of a seminal protein, sex peptide (SP), on the sex peptide receptor (SPR) in females. Moreover, we showed that the increased consumption of amino acids during the dark phase is induced in mated females independently of their internal level of amino acids. These results suggest that a post-mating SP/SPR signal elevates amino acid consumption during the dark phase <i>via</i> the circadian clock.</p></div

Free-running periods of the flies used in the CAFE assay.

<p>Free-running periods of the flies used in the CAFE assay.</p

Number of eggs laid in the light and dark phases differs for CS flies.

<p>The oviposition assay was performed for 12 h starting at either ZT 0 (for the light phase) or ZT 12 (for the dark phase). L and D represent results obtained during the light (orange bars) and dark (blue bars) phases, respectively; filled bars represent CS flies; hatched bars represent <i>per</i>⁰ flies. (A) The mean number of eggs laid during L and D phases on aa(-) medium by an amino acid-deprived female (n = 20). (B) The mean number of eggs laid during L and D phases on a standard cornmeal-agar-yeast-glucose medium (SM) by a female raised on SM (n = 20). Error bars indicate SEM. **<i>p</i> < 0.01 and ***<i>p</i> < 0.001 for comparisons between L and D phases using the Student’s <i>t</i>-test.</p

A post-mating signal elevates amino acid consumption during the dark phase.

<p>(A) The experimental scheme for the CAFE assays. Each L phase is shown by a white box and each D phase by a gray box. (B and C) Amino acid consumption during L (orange bars) and D (blue bars) phases was quantified using no-choice CAFE assays with the following strains: virgin CS females and CS females mated with CS or <i>SP</i>⁰/Δ¹³⁰ males (B; n = 3 or 4 trials); virgin <i>Df(1)Exel6234</i> (shown as Δ<i>SPR</i>) females and <i>Df(1)Exel6234</i> females mated with CS males (B; n = 4 trials); and virgin <i>ovo</i>^<i>D1</i>/CS females and <i>ovo</i>^<i>D1</i>/CS females mated with CS males (C; n = 4 trials). Intake per single fly is shown. Error bars indicate SEM. *<i>p</i> < 0.05 and **<i>p</i> < 0.01 for comparisons between L and D phases for each type of female in (B) and (C) using the Student’s <i>t</i>-test. <i>p</i> > 0.05 for all comparisons during L phase among females in (B) using one-way ANOVA. *<i>p</i> < 0.05 and **<i>p</i> < 0.01 for all comparisons during D phase among females in (B) using one-way ANOVA followed by <i>post hoc</i> Bonferroni/Dunn test. *<i>p</i> < 0.05 and **<i>p</i> < 0.01 for comparisons between <i>ovo</i>^<i>D1</i>/CS virgin and mated females in (C) using the Student’s <i>t</i>-test.</p

Mated <i>Drosophila melanogaster</i> females consume more amino acids during the dark phase

<div><p>To maintain homeostasis, animals must ingest appropriate quantities, determined by their internal nutritional state, of suitable nutrients. In the fruit fly <i>Drosophila melanogaster</i>, an amino acid deficit induces a specific appetite for amino acids and thus results in their increased consumption. Although multiple processes of physiology, metabolism, and behavior are under circadian control in many organisms, it is unclear whether the circadian clock also modulates such motivated behavior driven by an internal need. Differences in levels of amino acid consumption by flies between the light and dark phases of the day:night cycle were examined using a capillary feeder assay following amino acid deprivation. Female flies exhibited increased consumption of amino acids during the dark phase compared with the light phase. Investigation of mutants lacking a functional <i>period</i> gene (<i>per</i>⁰), a well-characterized clock gene in <i>Drosophila</i>, found no difference between the light and dark phases in amino acid consumption by <i>per</i>⁰ flies. Furthermore, increased consumption of amino acids during the dark phase was observed in mated but not in virgin females, which strongly suggested that mating is involved in the rhythmic modulation of amino acid intake. Egg production, which is induced by mating, did not affect the rhythmic change in amino acid consumption, although egg-laying behavior showed a <i>per</i>⁰-dependent change in rhythm. Elevated consumption of amino acids during the dark phase was partly induced by the action of a seminal protein, sex peptide (SP), on the sex peptide receptor (SPR) in females. Moreover, we showed that the increased consumption of amino acids during the dark phase is induced in mated females independently of their internal level of amino acids. These results suggest that a post-mating SP/SPR signal elevates amino acid consumption during the dark phase <i>via</i> the circadian clock.</p></div

Mated females increase amino acid consumption during the dark phase without amino acid deprivation.

<p>(A) The experimental scheme for the CAFE assays. Flies were tested without deprivation of amino acids. Each L phase is shown by a white box and each D phase by a gray box. (B) Amino acid consumption during L (orange bars) and D (blue bars) phases was quantified using two-choice CAFE assays between 50 mM glucose and 50 mM glucose containing 1/5 amino acid mixture in virgin and mated CS females (n = 6 trials). Intake of 50 mM glucose alone was subtracted from intake of 50 mM glucose containing 1/5 amino acid mixture, and the value was subsequently divided by the number of flies in each vial. The mean value is shown as “estimated amino acid intake”. Error bars indicate SEM. *<i>p</i> < 0.05 for comparisons between L and D phases and between virgin and mated females using the Student’s <i>t</i>-test.</p