SUMMARY 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-f, 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 GrLEXA/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 cDNA encoding a putative water channel protein, aquaporin, was cloned from a cDNA library of Aedes aegypti Malpighian tubules. The cDNA encodes a 26.11 kDa protein similar to insect aquaporins from Haematobia irritans exigua (Diptera) and Cicadella viridis (Homoptera), and to mammalian aquaporin 4. Localization of the messenger RNA (mRNA) was performed by in situ hybridization of Malpighian tubules and analysed by fluorescence and confocal microscopy. The mRNA was localized in tracheolar cells associated with the Malpighian tubules. No signal was detected in the Malpighian tubule epithelium. The molecular mechanisms for water movement between tissues and tracheoles are not yet elucidated in insects. Our results suggest a model to explain fluid movements in tracheoles during insect respiration.

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.

A cDNA cloned from Aedes aegypti (L.) (Aedae) female Malpighian tubule (AY596453) encodes a 584 amino acid residue protein (65.2 kDa) predicted as a G protein-coupled receptor and orthologue of the drosokinin receptor from Drosophila melanogaster and highly similar to the tick Boophilus microplus myokinin receptor (AF228521). Based on the similarity to this Aedes sequence, we also propose a correction for the Anopheles gambiae protein sequence EAA05450. When expressed in CHO-K1 cells, the Aedes receptor behaved as a multiligand receptor and functionally responded to concentrations > or = 1 nM of Aedae kinins 1-3, respectively, as determined by a calcium bioluminescence plate assay and single cell intracellular calcium measurements by confocal fluorescence cytometry. Estimates of EC50 values by the plate assay were 16.04 nM for Aedae-K-3, 26.6 nM for Aedae-K-2 and 48.8 nM for Aedae-K-1 and were statistically significantly different. These results suggest that the observed differences in physiological responses to the three Aedes kinins in the Aedes isolated Malpighian tubule reported elsewhere could now be explained by differences in intracellular signalling events triggered by the different peptides on the same receptor and not necessarily due to the existence of various receptors for the three Aedes kinins.

In the mosquito Aedes aegypti, 5-HT changes the endogenous rhythm of contractions in the female hindgut and increases fluid secretion in the larval Malpighian tubule. The role of 5-HT as a diuretic hormone in adults has been questioned. We cloned a cDNA encoding a serotonin receptor from a female A. aegypti Malpighian tubule library that is similar to the 5-HT7 receptor from Drosophila melanogaster. The transcript was localized in the tracheolar cells associated with the female Malpighian tubules but no signal was detectable in the tubule epithelium. Immunohistochemistry with specific antibodies confirmed the receptor expression in tracheolar cells and hindgut, and western blots of these tissues showed the expected 50 kDa band. The results suggest a role for serotonin in respiration and that this receptor may coordinate the tubule-hindgut response to serotonin during diuresis.

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.

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