The Molecular Characterization of a Diuretic Hormone Receptor (GPRdih1) From Females of the Yellow Fever Mosquito, Aedes aegypti (L.)

In the yellow fever mosquito, Aedes aegypti (L.), hemolymph-circulating diuretic hormones act upon the renal organs (Malpighian tubules) to regulate primary urine composition and secretion rate; however, the molecular endocrine mechanisms underlying rapid water elimination upon adult eclosion and blood feeding are not fully understood. Bioinformatic analysis of the current Aedes aegypti genome assembly reveals only a single predicted corticotropin releasing factor (CRF)-like diuretic hormone 44 (DH44) gene, but two DH44 receptor genes. The tissue expression profiles of the DH44 receptor(s), and specifically the identity of the DH44 receptor(s) in the Malpighian tubule, are undetermined in any mosquito species. This dissertation shows that Vectorbase gene ID AAEL008292 encodes a DH44 receptor (AaegGPRdih1) transcribed in Malpighian tubules. Sequence analysis and transcript localization indicate that AaegGPRdih1 is the co-ortholog of the Drosophila melanogaster DH44 receptor (CG12370-PA). The presence of conserved amino acid residues between AaegGPRdih1 and vertebrate CRF receptors suggests this mosquito receptor modulates multiple G protein-dependent intracellular signaling pathways. Quantitative PCR analysis of a time course of Malpighian tubule cDNA reveals AaegGPRdih1 abundance increases paralleling periods of observed urination. This suggests that target tissue receptor biology is linked to the known periods of release of diuretic hormones from the nervous system, pointing to a common up-stream regulatory mechanism. Higher relative abundance of AaegGPRdih1 transcript in female Malpighian tubules 24 hours after blood feeding suggests a role for AaegGPRdih1 in the excretion of nitrogen waste. RNA-mediated silencing to establish the significance of AaegGPRdih1 to mosquito Malpighian tubule physiology was inconclusive

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

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

<div><p>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 <i>Drosophila melanogaster</i>, a small subfamily of 8 <i>Gr</i> genes is thought to mediate the detection of sugars, the fly's major nutritional source. However, the specific roles for most <i>sugar Gr</i> genes are not known. Here, we report the generation of a series of mutant <i>sugar Gr</i> knock-in alleles and several composite <i>sugar Gr</i> mutant strains, including a sugar blind strain, which will facilitate the characterization of this gene family. Using Ca²⁺ imaging experiments, we show that most gustatory receptor neurons (GRNs) of sugar blind flies (lacking all 8 <i>sugar Gr</i> genes) fail to respond to any sugar tested. Moreover, expression of single <i>sugar Gr</i> genes in most sweet GRNs of sugar-blind flies does not restore sugar responses. However, when pair-wise combinations of <i>sugar Gr</i> genes are introduced to sweet GRNs, responses to select sugars are restored. We also examined the cellular phenotype of flies homozygous mutant for <i>Gr64a</i>, a <i>Gr</i> 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 <i>Gr64a</i> 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 <i>Gr</i> genes and construction of a sugar-blind strain provide powerful analytical tools for examining the roles of <i>Drosophila</i> and other insect <i>sugar Gr</i> genes in sweet taste.</p></div

Ribonucleosides are essential nutrients for rapid larval growth and survival.

<p>(a) Growth time in days from hatching of the first-instar larvae to eclosion (left) and survival rate (right) of larvae raised in different media shows that inosine and uridine are essential components. Larvae raised on HM grow slightly slower than, but have the same survival rate as, larvae raised on SCF. Replacing ribonucleosides with RNA (0.5 mg/mL) in HMΔ restores both growth time and survival rate, while replacing it with equimolar concentration of ribose fails to do so. Each bar represents the mean ± SEM (<i>n</i> = 4). Bars with different letters represent significant differences (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). Genotype: <i>w</i>^<i>1118</i>. The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s008" target="_blank">S4 Data</a>. (b) CaMPARI imaging of TO taste neurons shows that inosine and uridine, but none of the 3 other ribonucleosides, are potent ligands for Gr28 neurons. Uridine, cytidine (100 mM), and inosine (50 mM) were dissolved in water, while guanosine and adenosine were dissolved in DMSO and presented at concentration of 25 mM and 50 mM in water containing 25% and 10% f.c. DMSO, respectively. Each bar represents the mean ± SEM of ratios of red and green fluorescence intensities (<i>n</i> = 5–19). “*” represents significant differences between the preexposure (untreated) group to the groups with the indicated ligands applied (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). Genotype: <i>w</i>^<i>1118</i><i>; UAS-CaMPARI/Gr28a-GAL4</i>. The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s007" target="_blank">S3 Data</a>. (c) Two-choice preference assay shows that larvae require the <i>Gr28</i> genes to exhibit preference for uridine (50 mM, <i>n =</i> 12–24) and inosine (100 mM, <i>n</i> = 12–18). “*” represents significant difference between the genotypes (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). All the genotypes are compared to control. Genotypes: <i>w</i>^<i>1118</i>, <i>w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28 and w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28; genGr28/+</i>. The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s005" target="_blank">S1 Data</a>. f.c., final concentration; HM, holidic medium; SCF, standard cornmeal food; TO, terminal organ.</p

Larvae require <i>Gr28</i> genes for efficient growth and survival when presented with HM and HMΔ food.

<p>(a) About 40 eggs were deposited in 21-well microtiter plates containing 1 of 3 different foods: all wells containing HM (black; left), HMΔ (gray; middle), or a mixture of the two (12 HMΔ and 9 HM; right). Plates with either only HM or HMΔ medium were used to determine survival rate for complete (HM) or ribonucleoside-deficient (HMΔ) food. (b) Survival is displayed as percentage of flies hatched after eggs were deposited onto plate. For statistical analysis, survival in different foods was either compared across the same genotype (<i>Control</i> [black]: <i>w</i>^<i>1118</i>, Δ<i>Gr28</i> [red]: <i>w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28</i>, <i>and</i> Δ<i>Gr28 g-rescue</i> [green]: <i>w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28; genGr28/+</i>), or different genotypes were compared against the same mixed food (light–dark checkered pattern). Each bar represents the mean ± SEM (<i>n</i> = 5–6). Bars with different letters represent significant difference (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s008" target="_blank">S4 Data</a>. HM, holidic medium.</p

Genes of the Gr28 locus mediate larval taste preference for ribose, RNA, and arabinose.

<p>Two-choice feeding assays of wild-type and <i>Gr28</i> mutant larvae. (a) Larvae require the <i>Gr28</i> genes for taste preference for arabinose (<i>n =</i> 12–51), ribose (<i>n =</i> 12–36), and RNA (<i>n =</i> 21–36); Genotypes: <i>w</i>^<i>1118</i> (<i>Control</i>), <i>w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28</i> (Δ<i>Gr28</i>) <i>and</i> Δ<i>Gr28/</i>Δ<i>Gr28; Gr28 genomic rescue/</i>+ (Δ<i>Gr28 g-rescue</i>). (b) Single <i>Gr28</i> genes rescue taste preference for ribose in Δ<i>Gr28</i> homozygous mutant larvae. Genotypes were <i>w</i>^<i>1118</i> (lane 1), <i>w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28</i> (2), <i>w</i>^<i>1118</i>;Δ<i>Gr28/</i>Δ<i>Gr28; Gr28a-GAL4/+</i> (3), <i>w</i>^<i>1118</i>;Δ<i>Gr28/</i>Δ<i>Gr28; Gr28a-GAL4/UAS</i> (4, 6, 8, 10, 12, 14), and Δ<i>Gr28/</i>Δ<i>GrGr28; +/UAS</i> (5, 7, 9, 11, 13, 15) such that <i>UAS</i> represents indicated <i>transgene</i> (<i>n =</i> 12–36). (c) <i>Gr28</i> mutant larvae expressing a single <i>Gr28</i> gene in fructose-sensing (<i>Gr43a</i>^<i>GAL</i> -expressing) neurons show preference for ribose. Genotypes: <i>w</i>^<i>1118</i> (lane 1), <i>w</i>^<i>1118</i>; Δ<i>Gr28 Gr43</i>^<i>GAL4</i><i>/</i>Δ<i>Gr28 Gr43</i>^<i>GAL4</i> (2), and <i>w</i>^<i>1118</i>; Δ<i>Gr28 Gr43</i>^<i>GAL4</i><i>/</i>Δ<i>Gr28 Gr43</i>^<i>GAL4</i><i>; UAS/+</i>, such that <i>UAS</i> represent indicated transgene (<i>n =</i> 12–30). Each bar represents the mean ± SEM of two-choice preference responses. Concentrations were 100 mM (arabinose and ribose) or 0.5 mg/mL (RNA) in 1% agarose. Red “*” represents significant difference between indicated genotype and <i>w</i>^<i>1118</i> control (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). Green “*” represents significant difference between indicated genotype and Δ<i>Gr28 Gr43a</i>^<i>GAL4</i> double mutant (<i>w</i>^<i>1118</i>; Δ<i>Gr28 Gr43</i>^<i>GAL4</i><i>/</i>Δ<i>Gr28 Gr43</i>^<i>GAL4</i>). Two-tailed Mann-Whitney U test, <i>p</i> < 0.05). The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s005" target="_blank">S1 Data</a>.</p

RNA and ribose are ligands for single Gr28 proteins.

<p><b>(</b>a) Terminal taste neurons expressing the Ca²⁺ sensor CaMPARI require <i>Gr28a</i> in order to respond to ribose and RNA (<i>n =</i> 4–9). Genotypes: <i>w</i>^<i>1118</i><i>; UAS-CaMPARI/+; Gr28a-GAL4/+</i> (<i>control</i>), <i>w</i>^<i>1118</i>; Δ<i>Gr28 UAS-CaMPARI/</i>Δ<i>Gr28; Gr28a-Gal4/+</i> (ΔGr28), and <i>w</i>^<i>1118</i>; Δ<i>Gr28 UAS-CaMPARI /</i>Δ<i>Gr28; Gr28a-GAL4/UAS-Gr28a</i> (<i>Gr28a rescue</i>). (b) Expression of single <i>Gr28</i> genes conveys ribose and RNA responses to fructose-sensing pharyngeal taste neurons (<i>n =</i> 4–13). Genotypes: <i>w</i>^<i>1118</i><i>; UAS-CaMPARI Gr43a</i>^<i>GAL4</i><i>/+</i> (<i>control</i>), <i>w</i>^<i>1118</i><i>; Gr43</i>^<i>GAL4</i> <i>UAS-CaMPARI/+; UAS-Gr28a/+</i> (<i>Gr28a</i>), <i>w</i>^<i>1118</i><i>; Gr43</i>^<i>GAL4</i> <i>UAS-CaMPARI/+; UAS-Gr28b</i>.<i>a/+</i> (<i>Gr28b</i>.<i>a</i>), <i>w</i>^<i>1118</i><i>; Gr43</i>^<i>GAL4</i> <i>UAS-CaMPARI/+; UAS-Gr28b</i>.<i>e/+</i> (<i>Gr28b</i>.<i>e</i>). Final concentration of all substrates was 100 mM in water except for RNA (0.5 mg/mL). Representative images of the indicated genotypes are shown above the graphs. Scale bar is 10 μm. Each bar represents the mean ± SEM of ratios of red and green fluorescence intensities. “*” represents significant differences between the preexposure (no PC light, no chemical) group and a substrate group (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s007" target="_blank">S3 Data</a>. PC, photoconversion.</p

Larval preference for ribose and RNA is not mediated by sugar <i>Gr</i> genes.

<p><b>Two-choice preference assays for arabinose, ribose, deoxyribose, and RNA (panel a and c) and survival on these chemicals and nutritious sugars (panel b).</b> (a) Preference for arabinose is independent on various sugar <i>Gr</i> genes (<i>n</i> = 12–28). The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s005" target="_blank">S1 Data</a>. (b) Comparison of survival of <i>w</i>^<i>1118</i> larvae when kept on different substrates (<i>n</i> = 3–8). After 72 hours, approximately 50% of the larvae survive on agarose-only substrate (median survival, dashed line). For simplicity, significant differences are only indicated for median survival time. Data are represented as mean ± SEM. “*” represents significant difference between the larval survival on different substrates and agarose (two-tailed Mann-Whitney U test, <i>p</i> < 0.05). Reduced survival rate of larvae kept on arabinose and deoxyribose might be due to interference of these chemicals with sugar metabolism. The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s006" target="_blank">S2 Data</a>. (c) Larvae show strong preference for ribose (n = 12–36) and RNA (n = 6–36) when lacking <i>Gr43a</i> or the 8 <i>sGr</i>. Larvae are not attracted to deoxyribose (n = 6–24). As for fructose [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.ref004" target="_blank">4</a>], Δ<i>sGr</i> larvae showed stronger preference for ribose than wild-type larvae. Concentration of all substrates was 100 mM in 1% agarose, except RNA (0.5 mg/mL in 1% agarose). Genotypes: <i>w</i>^<i>1118</i> (control), <i>w</i>^<i>1118</i><i>; Gr43a</i>^<i>GAL4</i><i>/Gr43a</i>^{<i>GAL 4</i>}<i>(</i>Δ<i>Gr43a</i>), and <i>w</i>^<i>1118</i>; Δ<i>Gr61a</i> Δ<i>Gr64a-f/</i>Δ<i>Gr61a</i> Δ<i>Gr64a-f (</i>Δ<i>sGrs</i>). The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s005" target="_blank">S1 Data</a>. Gr, gustatory receptor; PREF, preference index; <i>sGr</i>, sugar <i>Gr</i> gene.</p

Expression of the 6 <i>Gr28</i> genes in third-instar larvae.

<p>(a) Graphic summary of <i>Gr28</i> gene expression. Cells and neurons with their axons expressing the respective GAL4 driver are all shown in green. Brain is shown in grey, and the digestive system—including the pharynx, PV, and gut—are outlined. (b) Live GFP imaging of the larval head, showing expression of 3 genes (<i>Gr28a</i>, <i>Gr28b.a</i>, and <i>Gr28b.e</i>) in neurons of the TO. <i>Gr28b.d</i> is expressed in neurons of the DPS and VPS organs, while <i>Gr28a</i> is also expressed in the PPS organ. Neither <i>Gr28a</i> nor <i>Gr28b.d</i> are co-expressed with <i>Gr43^GAL4</i> (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s002" target="_blank">S1 Fig</a>). Number of larvae with GFP positive taste neurons/total number of larvae analyzed were 7/7 for <i>Gr28a</i>, 4/4 for <i>Gr28b.a</i>, 0/5 for <i>Gr28b.b</i>, 0/7 for <i>Gr28b.c</i>, 5/5 for <i>Gr28b.d</i>, and 5/5 for <i>Gr28b.e</i>. (c) View of the brain and parts of the ventral nerve cord, showing different degrees of expression for each of the 6 <i>Gr28</i> genes. The brains were stained with anti-GFP antibody (green) and counterstained with nc82 antibody (red). Number of larvae with GFP antibody–positive staining in the brain-VNC/number of brains analyzed were 3/3 for <i>Gr28a</i>, 5/5 for <i>Gr28b.a</i>, 3/3 for <i>Gr28b.b</i>, 5/5 for <i>Gr28b.c</i>, 3/3 for <i>Gr28b.d</i>, and 6/6 for <i>Gr28b.e</i>. (d) Live GFP imaging of the PV and midgut, showing expression of all <i>Gr28</i> genes with the exception of <i>Gr28b.d</i>. Expression of <i>Gr28b.a</i> and <i>Gr28b.e</i> is broad and includes the PV and midgut, while expression of <i>Gr28a</i> and <i>Gr28b.b</i> is defined to a smaller area of the gut only. Number of larvae with GFP-positive cells/total number of larvae analyzed were 5/5 for <i>Gr28a</i>, 4/4 for <i>Gr28b.a</i>, 3/3 for <i>Gr28b.b</i>, 5/5 for <i>Gr28b.c</i>, 0/5 for <i>Gr28b.d</i>, and 4/4 for <i>Gr28b.e</i>. (e) Summary of tissues expressing each of the 6 <i>Gr28</i> genes. Genotypes were <i>w</i>; <i>UAS-mCD8GFP/Gr28x-GAL4</i>, such that x refers to indicated <i>Gr-Gal4</i> driver. Scale bar is 100 μm. For live imaging (panel b and d), at least 5 larvae for each genotype were analyzed, and GFP cells in taste sensilla and the gut were observed in each case for <i>Gr28a</i>, <i>Gr28ba</i>, <i>Gr28b.e</i>, <i>Gr28b.d</i>, and <i>Gr28b.e</i>; for staining (panel c), at least 3 brains for each genotype were analyzed, with GFP-positive neurons observed in each case. The images are good representatives of these experiments. DPS, dorsal pharyngeal sensory; GFP, green fluorescent protein; <i>Gr28</i>, gustatory receptor subfamily 28; PPS, posterior pharyngeal sensory; PV, proventriculus; TO, terminal organ; VPS, ventral pharyngeal sensory.</p