RNA Taste Is Conserved in Dipteran Insects.

BACKGROUND: Ribonucleosides and RNA are an underappreciated nutrient group essential during Drosophila larval development and growth. Detection of these nutrients requires at least one of the 6 closely related taste receptors encoded by the Gr28 genes, one of the most conserved insect taste receptor subfamilies. OBJECTIVES: We investigated whether blow fly larvae and mosquito larvae, which shared the last ancestor with Drosophila about 65 and 260 million years ago, respectively, can taste RNA and ribose. We also tested whether the Gr28 homologous genes of the mosquitoes Aedes aegypti and Anopheles gambiae can sense these nutrients when expressed in transgenic Drosophila larvae. METHODS: Taste preference in blow flies was examined by adapting a 2-choice preference assay that has been well-established for Drosophila larvae. For the mosquito Aedes aegypti, we developed a new 2-choice preference assay that accommodates the aquatic environment of these insect larvae. Finally, we identified Gr28 homologs in these species and expressed them in Drosophila melanogaster to determine their potential function as RNA receptors. RESULTS: Larvae of the blow fly Cochliomyia macellaria and Lucilia cuprina are strongly attracted to RNA (0.5 mg/mL) in the 2-choice feeding assays (P < 0.05). Similarly, the mosquito Aedes aegypti larvae showed a strong preference for RNA (2.5 mg/mL) in an aquatic 2-choice feeding assay. Moreover, when Gr28 homologs of Aedes or Anopheles mosquitoes are expressed in appetitive taste neurons of Drosophila melanogaster larvae lacking their Gr28 genes, preference for RNA (0.5 mg/mL) and ribose (0.1 M) is rescued (P < 0.05). CONCLUSIONS: The appetitive taste for RNA and ribonucleosides in insects emerged about 260 million years ago, the time mosquitoes and fruit flies diverged from their last common ancestor. Like sugar receptors, receptors for RNA have been highly conserved during insect evolution, suggesting that RNA is a critical nutrient for fast-growing insect larvae.

The taste of ribonucleosides: Novel macronutrients essential for larval growth are sensed by Drosophila gustatory receptor proteins.

Animals employ various types of taste receptors to identify and discriminate between different nutritious food chemicals. These macronutrients are thought to fall into 3 major groups: carbohydrates/sugars, proteins/amino acids, and fats. Here, we report that Drosophila larvae exhibit a novel appetitive feeding behavior towards ribose, ribonucleosides, and RNA. We identified members of the gustatory receptor (Gr) subfamily 28 (Gr28), expressed in both external and internal chemosensory neurons as molecular receptors necessary for cellular and appetitive behavioral responses to ribonucleosides and RNA. Specifically, behavioral preference assays show that larvae are strongly attracted to ribose- or RNA-containing agarose in a Gr28-dependent manner. Moreover, Ca2+ imaging experiments reveal that Gr28a-expressing taste neurons are activated by ribose, RNA and some ribonucleosides and that these responses can be conveyed to Gr43aGAL4 fructose-sensing neurons by expressing single members of the Gr28 gene family. Lastly, we establish a critical role in behavioral fitness for the Gr28 genes by showing that Gr28 mutant larvae exhibit low survival rates when challenged to find ribonucleosides in food. Together, our work identifies a novel taste modality dedicated to the detection of RNA and ribonucleosides, nutrients that are essential for survival during the accelerated growth phase of Drosophila larvae.

Drosophila sugar receptors in sweet taste perception, olfaction, and internal nutrient sensing.

Identification of nutritious compounds is dependent on expression of specific taste receptors in appropriate taste-cell types [1]. In contrast to mammals, which rely on a single, broadly tuned heterodimeric sugar receptor [2], the Drosophila genome harbors a small subfamily of eight, closely related gustatory receptor (Gr) genes, Gr5a, Gr61a, and Gr64a-Gr64f, of which three have been proposed to mediate sweet taste [3-6]. However, expression and function of several of these putative sugar Gr genes are not known. Here, we present a comprehensive expression and functional analysis using Gr(LEXA/GAL4) alleles that were generated through homologous recombination. We show that sugar Gr genes are expressed in a combinatorial manner to yield at least eight sets of sweet-sensing neurons. Behavioral investigations show that most sugar Gr mutations affect taste responses to only a small number of sugars and that effective detection of most sugars is dependent on more than one Gr gene. Surprisingly, Gr64a, one of three Gr genes previously proposed to play a major role in sweet taste [3, 4], is not expressed in labellar taste neurons, and Gr64a mutant flies exhibit normal sugar responses elicited from the labellum. Our analysis provides a molecular rationale for distinct tuning profiles of sweet taste neurons, and it favors a model whereby all sugar Grs contribute to sweet taste. Furthermore, expression in olfactory organs and the brain implies novel roles for sugar Gr genes in olfaction and internal nutrient sensing, respectively. Thus, sugar receptors may contribute to feeding behavior via multiple sensory systems.

A genetic tool kit for cellular and behavioral analyses of insect sugar receptors.

Arthropods employ a large family of up to 100 putative taste or gustatory receptors (Grs) for the recognition of a wide range of non-volatile chemicals. In Drosophila melanogaster, a small subfamily of 8 Gr genes is thought to mediate the detection of sugars, the fly's major nutritional source. However, the specific roles for most sugar Gr genes are not known. Here, we report the generation of a series of mutant sugar Gr knock-in alleles and several composite sugar Gr mutant strains, including a sugar blind strain, which will facilitate the characterization of this gene family. Using Ca(2+) imaging experiments, we show that most gustatory receptor neurons (GRNs) of sugar blind flies (lacking all 8 sugar Gr genes) fail to respond to any sugar tested. Moreover, expression of single sugar Gr genes in most sweet GRNs of sugar-blind flies does not restore sugar responses. However, when pair-wise combinations of sugar Gr genes are introduced to sweet GRNs, responses to select sugars are restored. We also examined the cellular phenotype of flies homozygous mutant for Gr64a, a Gr gene previously reported to be a major contributor for the detection of many sugars. In contrast to these claims, we find that sweet GRNs of Gr64a homozygous mutant flies show normal responses to most sugars, and only modestly reduced responses to maltose and maltotriose. Thus, the precisely engineered genetic mutations of single Gr genes and construction of a sugar-blind strain provide powerful analytical tools for examining the roles of Drosophila and other insect sugar Gr genes in sweet taste.