Sugar receptors in Drosophila

Abstract

The detection and discrimination of chemical compounds in potential foods are essential sensory processes when animals feed. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of mostly nonvolatile chemicals that include sugars, a diverse group of toxic compounds present in many inedible plants and spoiled foods, and pheromones [1-6]. With the exception of a trehalose (GR5a) and a caffeine (GR66a) receptor [7-9], the functions of GRs involved in feeding are unknown. Here, we show that the Gr64 genes encode receptors for numerous sugars. We generated a fly strain that contained a deletion for all six Gr64 genes (DeltaGr64) and showed that these flies exhibit no or a significantly diminished proboscis extension reflex (PER) response when stimulated with glucose, maltose, sucrose, and several other sugars. The only considerable response was detected when Gr64 mutant flies were stimulated with fructose. Interestingly, response to trehalose is also abolished in these flies, even though they contain a functional Gr5a gene, which has been previously shown to encode a receptor for this sugar [8, 9]. This observation indicates that two or more Gr genes are necessary for trehalose detection, suggesting that GRs function as multimeric receptor complexes. Finally, we present evidence that some members of the Gr64 gene family are transcribed as a polycistronic mRNA, providing a mechanism for the coexpression of multiple sugar receptors in the same taste neurons.

Figure 1. Generation of a Gr64 mutant strain (ΔGr64) and RT-PCR expression analysis of the six Gr64 genes

(A) Diagram of the Gr64 gene cluster. The positions of the piggyBac transposons are indicated by triangles. The diagram shows the Gr64 cluster prior to generation of the deletion line. The numbered arrows indicate the positions of the primers used for PCR analysis of the deletion. The black bars represent the rescue constructs for the genes flanking the Gr64 cluster. (B) Molecular analysis of ΔGr64 mutant strain. The diagram shows the structure of the Gr64 deletion (ΔGr64) after trans-recombination. Genomic DNA from w¹¹¹⁸ flies was also analyzed for comparison. Expected band sizes are as follows: 1.1 kb for primers T1 and T2, 1.5 kb for primers 11 and 12, and 6.9 kb for primers 13 and 14. Relevant band sizes from the ladder are marked along the sides of the gel. The 1.5 and 6.9 kb products were cloned and sequenced to further confirm the presence of the deletion. The 1.1 kb product is derived from the tubulin gene and serves as a control for DNA integrity. (C) Exon-intron structure of the Gr64 cluster. Exons are represented by boxes and introns by v-shaped lines. The numbered arrows show the positions of the primers used for RT-PCR analysis. The black bar indicates the rescue construct (UAS-Gr64abcd_GFP_f) in which Gr64e was replaced by EGFP (indicated by the dashed line). (D) RT-PCR of total RNA from fly heads indicates the presence of polycistronic transcripts in the Gr64 cluster. RNA was extracted from wild-type ORE-R flies. Each pair of primers spans at least one intron in each of the two genes being investigated. For each pair of primers used in an RT-PCR reaction, a corresponding PCR reaction was performed on genomic DNA to provide a size comparison. RT-PCR products were isolated for each primer pair, cloned and sequenced to confirm integrity of appropriately spliced cDNA products. RT-PCR from leg tissue showed similar results (data not shown). Lanes marked “RT” represent RT-PCR products, while lanes marked “G” represent PCR products from genomic DNA.

Figure 2. ΔGr64 mutants are severely deficient in the perception of most sugars Proboscis Extension Response (PER) of ΔGr64 mutants and isogenic control flies to 500mM (A) and 100mM (B) sugar solutions

The genotype of ΔGr64 mutants is R1/+;R2/+;ΔGr64/ΔGr64 and that of the control flies is f03449/d06001. “Probability of Extension” represents the number of times flies from a given strain extended their proboscis when presented with a tastant, divided by the total number of times that the tastant was presented. For all data shown in Figure 2, each graph is the average of 4–15 experiments +/− SEM (3–11 flies per experiment, 20–105 flies total for each strain and tastant tested). Asterisks indicate a significant difference between the mutant and control strains, as determined by Student’s t-test (* indicates p<0.05, *** indicates p<0.0001). Glycerol was used as 10% or 2% solutions in water. (C) The Gr5a gene is functional in R1/Y;R2/+;ΔGr64/ΔGr64 flies. Flies heterozygous for ΔGr64, but containing the same X chromosome (i.e. the same Gr5a) as the homozygous ΔGr64 flies, show normal and robust response to trehalose at both 100 mM and 500 mM concentrations. (D) PER response of ΔGr64 mutant and control strains to various bitter tastants in the presence of 500mM fructose. The response to 500mM fructose alone is shown for comparison. There was no significant difference between mutants and controls for any of the bitter solutions by Student’s t-test.

Figure 3. Rescue of ΔGr64 mutant phenotype

PER response of ΔGr64 mutants (R1/+;R2/+;ΔGr64/ΔGr64) carrying one copy of the UAS-Gr64abcd_GFP_f reporter, with or without Gr5a-Gal4 driver. Sugars were tested at 500mM (A) and 100mM (B) concentration. PER response of flies with the rescue construct is similar to that of the control flies, regardless of whether or not the Gr5a-Gal4 driver is present. Each graph is the average of 4–15 experiments +/− SEM (3–11 flies per experiment, 20–105 flies total for each strain and tastant tested). Asterisks indicate a significant difference between the mutant and control strains, as determined by Student’s t-test (* indicates p<0.05, ** indicates p<0.001, *** indicates p<0.0001).