Identification of additional dye tracers for measuring solid food intake and food preference via consumption-excretion in Drosophila.

The Drosophila model has become a leading platform for investigating mechanisms that drive feeding behavior and the effect of diet on physiological outputs. Several methods for tracking feeding behavior in flies have been developed. One method, consumption-excretion or Con-Ex, provides flies with media labeled with dye and then quantifies the amount of dye excreted into the vial as a measure of consumption. We previously found that Blue 1 and Orange 4 work well in Con-Ex and can be used as a dye pair in food preference studies. We have expanded our development of Con-Ex by identifying two additional dyes, Orange G and Yellow 10, that detect the anticipated effects of mating status, strain, starvation and nutrient concentration. Additionally, Orange G and Yellow 10 accumulate linearly in excretion products out to 48 h and the excreted volumes of these two dyes reflect the volumes consumed. Orange G also works with Blue 1 as a dye pair in food preference studies. Finally, consumption of Blue 1, Orange 4, Orange G or Yellow 10 does not affect ethanol sedation or rapid tolerance to ethanol. Our findings establish that Orange G and Yellow 10, like Blue 1 and Orange 4, are suitable for use in Con-Ex.

Expansion and application of dye tracers for measuring solid food intake and food preference in Drosophila.

The Drosophila model is used to investigate the effects of diet on physiology as well as the effects of genetic pathways, neural systems and environment on feeding behavior. We previously showed that Blue 1 works well as a dye tracer to track consumption of agar-based media in Drosophila in a method called Con-Ex. Here, we describe Orange 4 as a novel dye for use in Con-Ex studies that expands the utility of this method. Con-Ex experiments using Orange 4 detect the predicted effects of starvation, mating status, strain, and sex on feeding behavior in flies. Orange 4 is consumed and excreted into vials linearly with time in Con-Ex experiments, the number of replicates required to detect differences between groups when using Orange 4 is comparable to that for Blue 1, and excretion of the dye reflects the volume of consumed dye. In food preference studies using Orange 4 and Blue 1 as a dye pair, flies decreased their intake of food laced with the aversive tastants caffeine and NaCl as determined using Con-Ex or a more recently described modification called EX-Q. Our results indicate that Orange 4 is suitable for Con-Ex experiments, has comparable utility to Blue 1 in Con-Ex studies, and can be paired with Blue 1 to assess food preference via both Con-Ex and EX-Q.

Dietary yeast influences ethanol sedation in Drosophila via serotonergic neuron function.

Abuse of alcohol is a major clinical problem with far-reaching health consequences. Understanding the environmental and genetic factors that contribute to alcohol-related behaviors is a potential gateway for developing novel therapeutic approaches for patients that abuse the drug. To this end, we have used Drosophila melanogaster as a model to investigate the effect of diet, an environmental factor, on ethanol sedation. Providing flies with diets high in yeast, a routinely used component of fly media, increased their resistance to ethanol sedation. The yeast-induced resistance to ethanol sedation occurred in several different genetic backgrounds, was observed in males and females, was elicited by yeast from different sources, was readily reversible, and was associated with increased nutrient intake as well as decreased internal ethanol levels. Inhibition of serotonergic neuron function using multiple independent genetic manipulations blocked the effect of yeast supplementation on ethanol sedation, nutrient intake, and internal ethanol levels. Our results demonstrate that yeast is a critical dietary component that influences ethanol sedation in flies and that serotonergic signaling is required for the effect of dietary yeast on nutrient intake, ethanol uptake/elimination, and ethanol sedation. Our studies establish the fly as a model for diet-induced changes in ethanol sedation and raise the possibility that serotonin might mediate the effect of diet on alcohol-related behavior in other species.

Convergent Evidence From Humans and Drosophila melanogaster Implicates the Transcription Factor MEF2B/Mef2 in Alcohol Sensitivity.

BACKGROUND: Self-Rating of the Effects of Alcohol (SRE) measures level of response to ethanol (EtOH) in humans. Interestingly, there is a positive relationship between the SRE and risk for abusing alcohol, suggesting mechanistic connections between SRE and alcohol abuse. METHODS: To identify candidate genes with a role in SRE and alcohol-related behavior more generally, we coupled human genetic analyses with studies in Drosophila melanogaster. We first performed a gene-based analysis of Genomewide association studies (GWAS) summary statistics for SRE in the Avon Longitudinal Study of Parents and Children sample. Based on prior findings in humans, orthology to fly genes, and the availability of genetic reagents, we selected a subset of these genes for studies on EtOH behavior in Drosophila. RESULTS: We found 37 genes with nominal associations in our SRE GWAS. We explored the role of 6 orthologous genes in Drosophila EtOH sedation and rapid tolerance. We found that the transcription factor Mef2 is required for normal EtOH sedation in flies. Pan-neuronal expression of 2 independent Mef2 RNAi transgenes significantly reduced Mef2 expression and made flies resistant to EtOH sedation. Additionally, flies with multiple independent mutant alleles of Mef2 were also resistant to EtOH sedation, confirming a role for Mef2 in this behavior. Altered expression of Mef2 did not change EtOH rapid tolerance or cause a net change in internal EtOH concentrations. CONCLUSIONS: Our studies indicate that MEF2B influences SRE in humans and that Mef2 impacts EtOH sedation in Drosophila.

Measurement of solid food intake in Drosophila via consumption-excretion of a dye tracer.

Although the Drosophila melanogaster (fly) model is a popular platform for investigating diet-related phenomena, it can be challenging to measure the volume of agar-based food media flies consume. We addressed this challenge by developing a dye-based method called Consumption-Excretion (Con-Ex). In Con-Ex studies, flies consume solid food labeled with dye, and the volume of food consumed is reflected by the sum of the dye inside of and excreted by flies. Flies consumed-excreted measurable amounts of FD&C Blue No. 1 (Blue 1) and other dyes in Con-Ex studies, but only Blue 1 was readily detectable at concentrations that had no discernable effect on consumption-excretion. In studies with Blue 1, consumption-excretion (i) increased linearly with feeding duration out to 24 h at two different laboratory sites, (ii) was sensitive to starvation, mating status and strain, and (iii) changed in response to alteration of media composition as expected. Additionally, the volume of liquid Blue 1 consumed from capillary tubes was indistinguishable from the volume of Blue 1 excreted by flies, indicating that excreted Blue 1 reflects consumed Blue 1. Our results demonstrate that Con-Ex with Blue 1 as a food tracer is a useful method for assessing ingestion of agar-based food media in adult flies.