Detection of Organic Acids by the Taste Systems in Drosophila melanogaster and Drosophila sechellia

In this work we examine the response of two Drosophila species to acids found in their natural food sources. We show that acids reduce normal gustatory behavior to sweet stimuli. We find that this acid aversion behavior is mediated by both the sweet and bitter neurons in the Drosophila taste system. Through electrophysiological studies we determine that acids inhibit neuronal firing to sucrose independently of bitter neuron input. We show that this inhibition can be overcome by increased sucrose concentration. We also find that acid inhibition of the sweet neuron is dependent on pH, regardless of the anion of the acid. Lastly, we examined the behavior of two members of the melanogaster subgroup, Drosophila sechellia and Drosophila melanogaster, to acids present in the main food source of Drosophila sechellia, morinda fruit. Behavioral studies showed that these two species diverge in their responses to morinda fruit and its component acids. Upon further examination we find that for some of the acids the behavioral difference can be attributed to a reduced sweet neuron inhibition in Drosophila sechellia. Thus we propose that sweet neuron inhibition plays an important role in behavior towards acidic stimuli

Detection of Organic Acids by the Taste Systems in Drosophila melanogaster and Drosophila sechellia

In this work we examine the response of two Drosophila species to acids found in their natural food sources. We show that acids reduce normal gustatory behavior to sweet stimuli. We find that this acid aversion behavior is mediated by both the sweet and bitter neurons in the Drosophila taste system. Through electrophysiological studies we determine that acids inhibit neuronal firing to sucrose independently of bitter neuron input. We show that this inhibition can be overcome by increased sucrose concentration. We also find that acid inhibition of the sweet neuron is dependent on pH, regardless of the anion of the acid. Lastly, we examined the behavior of two members of the melanogaster subgroup, Drosophila sechellia and Drosophila melanogaster, to acids present in the main food source of Drosophila sechellia, morinda fruit. Behavioral studies showed that these two species diverge in their responses to morinda fruit and its component acids. Upon further examination we find that for some of the acids the behavioral difference can be attributed to a reduced sweet neuron inhibition in Drosophila sechellia. Thus we propose that sweet neuron inhibition plays an important role in behavior towards acidic stimuli

Evolution of fatty acid taste in drosophilids.

Comparative studies of related but ecologically distinct species can reveal how the nervous system evolves to drive behaviors that are particularly suited to certain environments. Drosophila melanogaster is a generalist that feeds and oviposits on most overripe fruits. A sibling species, D. sechellia, is an obligate specialist of Morinda citrifolia (noni) fruit, which is rich in fatty acids (FAs). To understand evolution of noni taste preference, we characterized behavioral and cellular responses to noni-associated FAs in three related drosophilids. We find that mixtures of sugar and noni FAs evoke strong aversion in the generalist species but not in D. sechellia. Surveys of taste sensory responses reveal noni FA- and species-specific differences in at least two mechanisms-bitter neuron activation and sweet neuron inhibition-that correlate with shifts in noni preference. Chemoreceptor mutant analysis in D. melanogaster predicts that multiple genetic changes account for evolution of gustatory preference in D. sechellia

The Drosophila melanogaster Phospholipid Flippase dATP8B Is Required for Odorant Receptor Function

The olfactory systems of insects are fundamental to all aspects of their behaviour, and insect olfactory receptor neurons (ORNs) exhibit exquisite specificity and sensitivity to a wide range of environmental cues. In Drosophila melanogaster, ORN responses are determined by three different receptor families, the odorant (Or), ionotropic-like (IR) and gustatory (Gr) receptors. However, the precise mechanisms of signalling by these different receptor families are not fully understood. Here we report the unexpected finding that the type 4 P-type ATPase phospholipid transporter dATP8B, the homologue of a protein associated with intrahepatic cholestasis and hearing loss in humans, is crucial for Drosophila olfactory responses. Mutations in dATP8B severely attenuate sensitivity of odorant detection specifically in Or-expressing ORNs, but do not affect responses mediated by IR or Gr receptors. Accordingly, we find dATP8B to be expressed in ORNs and localised to the dendritic membrane of the olfactory neurons where signal transduction occurs. Localisation of Or proteins to the dendrites is unaffected in dATP8B mutants, as is dendrite morphology, suggesting instead that dATP8B is critical for Or signalling. As dATP8B is a member of the phospholipid flippase family of ATPases, which function to determine asymmetry in phospholipid composition between the outer and inner leaflets of plasma membranes, our findings suggest a requirement for phospholipid asymmetry in the signalling of a specific family of chemoreceptor proteins

The Drosophila melanogaster phospholipid flippase dATP8B is required for odorant receptor function.

The olfactory systems of insects are fundamental to all aspects of their behaviour, and insect olfactory receptor neurons (ORNs) exhibit exquisite specificity and sensitivity to a wide range of environmental cues. In Drosophila melanogaster, ORN responses are determined by three different receptor families, the odorant (Or), ionotropic-like (IR) and gustatory (Gr) receptors. However, the precise mechanisms of signalling by these different receptor families are not fully understood. Here we report the unexpected finding that the type 4 P-type ATPase phospholipid transporter dATP8B, the homologue of a protein associated with intrahepatic cholestasis and hearing loss in humans, is crucial for Drosophila olfactory responses. Mutations in dATP8B severely attenuate sensitivity of odorant detection specifically in Or-expressing ORNs, but do not affect responses mediated by IR or Gr receptors. Accordingly, we find dATP8B to be expressed in ORNs and localised to the dendritic membrane of the olfactory neurons where signal transduction occurs. Localisation of Or proteins to the dendrites is unaffected in dATP8B mutants, as is dendrite morphology, suggesting instead that dATP8B is critical for Or signalling. As dATP8B is a member of the phospholipid flippase family of ATPases, which function to determine asymmetry in phospholipid composition between the outer and inner leaflets of plasma membranes, our findings suggest a requirement for phospholipid asymmetry in the signalling of a specific family of chemoreceptor proteins

Mutations in <i>dATP8B</i> cause severe olfactory defects.

<p>(A) Electroantennogram (EAG) and (B) electropalpogram (EPG) responses to a panel of odorants. Bars represent mean response ± SEM (n = 10), asterisks are significant differences. Responses of homozygous <i>ll2</i> mutant flies (blue bars) are severely reduced when compared with controls (black bars) for all the tested odorants except CO₂ (t-test, Bonferroni, <i>p&lt;0.05</i>). Heterozygote flies (grey bars) are not affected. (C) A deletion that removes <i>dATP8B</i> and a <i>piggyBac</i> element inserted in <i>dATP8B</i> both fail to complement the <i>ll2</i> phenotype. Bars represent mean EAG responses ± SEM (n = 6–10). Trans-heterozygotes for <i>ll2</i> and deficiency <i>Df(3R)Exel8155</i> (blue bars) have reduced EAG response compared to controls (black bars, t-tests, Bonferroni, p&lt;0.05), and homozygotes for the <i>piggyBac</i> insertion <i>dATP8B^f05203</i> (red bars) and trans-heterozygotes for <i>ll2</i> and <i>dATP8B^f05203</i> (red/blue hatch) show similar reductions when compared to heterozygote controls (grey bars; t-tests, Bonferroni, p&lt;0.05). (D) The mapped genomic interval for the <i>ll2</i> mutant and the gene model of <i>dATP8B (CG14741)</i>. The candidate region contained 14 annotated genes. The identified nonsense mutation in <i>dATP8B</i> and the insertion site of the <i>piggyBac</i> line <i>dATP8B^f05203</i> are in the 1^st and the 10^th common coding exon respectively, affecting all the annotated transcripts. For isoforms RC, RF, RG, RH and RI the EMS mutation causes R18-X and for isoforms RB and RD the mutation causes R197-X. Coding exons are colored in orange and the 3′ and the 5′ UTR are in grey. (E) The olfactory defect in the <i>dATP8B^f05203</i> line is reverted when the <i>piggyBac</i> insertion is precisely excised. EAG responses of homozygous <i>dATP8B^f05203-Ex</i> flies (red bars) to a panel of odorants were not different from controls (black bars). Bars represent mean response ± SEM (n = 5, t-test, Bonferroni). Odorants are: EA, ethyl acetate, PA, pentyl acetate, MS, methyl salicylate, OL, 1-octen-3-ol, HB, ethyl 3-hydroxybutanoate, EH, ethyl hexanoate, MH, 6-methyl-5-hepten-2-one, BZ, benzaldehyde, MP, 4-methylphenol, PO, paraffin oil (solvent blank).</p

Orco and Or22a localize normally to the dendrites in <i>dATP8B</i> mutants.

<p>14 µm thick antennal sections from wild type flies (CS-5) were stained for anti-Orco or for anti-Or22a. No difference in either Orco or Or22a localisation to the outer dendrites was observed in <i>dATP8B</i> mutants (<i>dATP8B^f05203</i>) compared to control flies.</p

Orco and Or22a localize normally to the dendrites in <i>dATP8B</i> mutants.

<p>14 µm thick antennal sections from wild type flies (CS-5) were stained for anti-Orco or for anti-Or22a. No difference in either Orco or Or22a localisation to the outer dendrites was observed in <i>dATP8B</i> mutants (<i>dATP8B^f05203</i>) compared to control flies.</p

<i>dATP8B</i> is expressed and required in <i>Or-</i>expressing olfactory receptor neurons.

<p>(A–A′) dATP8B protein localises to the dendrites of ORNs. 14 µm thick antennal sections from wild type flies (CS-5) were stained for anti-dATP8B (magenta). Strong anti-dATP8B staining is seen in the shafts of the sensilla (the location of the outer dendrites) of basiconic (arrows) and trichoid (arrowhead) sensilla. Staining is also seen in the inner dendrites and cell bodies, however this staining is also present in <i>dATP8B</i> mutants. (B) The strong outer dendrite staining of anti-dATP8B is absent in <i>dATP8B</i> mutants (<i>ll2</i>/<i>Df(3R)Exel8155</i>), indicating it is specific for dATP8B. (C) dATP8B and Orco co-localise in the outer dendrites. 14 µm thick antennal sections from wild type flies were stained for anti-dATP8B (magenta) and Orco (green). (D) dATP8B is absent from the outer dendrites of <i>Gr21a</i>-expressing (ab1C) neurons. 14 µm thick antennal sections from Gr21a&gt;mCD8:GFP flies were stained for anti-dATP8B (magenta) and anti-GFP (green). (E) In <i>Orco-GAL4: UAS-dATP8B^RNAi</i> flies (red bars) the EAG response is significantly reduced compared to controls (black and grey bars, t-test, Bonferroni, <i>p&lt;0.05</i>) for some of the same odorants that are affected by the two <i>dATP8B</i> mutant alleles. Bars represent mean EAG responses ± SEM (n = 6–10). Odorants are: EA, ethyl acetate, PA, pentyl acetate, MS, methyl salicylate, OL, 1-octen-3-ol, HB, ethyl 3-hydroxybutanoate, EH, ethyl hexanoate, BZ, benzaldehyde, PO, paraffin oil (solvent blank).</p

<i>dATP8B</i> is required for responses of <i>Or</i> but not <i>IR</i> or <i>Gr-</i> expressing sensory neurons.

<p>(A–C) Mutations in <i>dATP8B</i> reduce the sensitivity of <i>Or</i>-expressing neurons to their major ligands. Dose-response curves for neurons located in three different morphological types of sensilla are shown; (A) ab3B neurons in basiconic sensilla (<i>Or85b</i>) to 2-heptanone, (B) at1A neurons in trichoid sensilla (<i>Or67d</i>) to cis-vaccenyl acetate, (C) ac3B neurons in coeloconic sensilla (<i>Or35a</i>) to Z3-hexenol. In all cases sensitivity is significantly lowered for all doses in homozygous <i>dATP8B^f05203</i> flies (red) compared to controls (black, t-test, Bonferroni, n = 6–9, recorded from 4–8 flies). (D–E) Mutations in <i>dATP8B</i> differentially alter responses of ab3A neurons (<i>Or22a</i>) to a major ligand ethyl hexanoate (D) and a minor ligand ethyl butanoate (E). (n = 7 sensilla, recorded from 5–6 flies). (F–I) Neurons expressing <i>IR</i> or <i>Gr</i> receptors are not affected by <i>dATP8B</i> mutations. Four different types of neurons are shown; (F) responses of ac2A neurons in coeloconic sensilla (<i>IR41a</i> and <i>IR76b</i>) to 1,4-diaminobutane, (G) responses of ac3A neurons in coeloconic sensilla (<i>IR75a,b and c</i>) to propionic acid, (H) responses of ab1C neurons in basiconic sensilla (<i>Grs21a</i> and <i>63a</i>) to CO₂, (I) responses from single neurons in labellar taste sensilla expressing <i>Gr</i> genes to 100 mM sucrose (Suc) or 10 mM caffeine (Caff) (mean ± SEM). In all cases control and mutant responses are not significantly different (n = 6–10 sensilla from 3–5 flies, t-tests, Bonferroni). Controls are either wild type or heterozygous <i>dATP8B^f05203</i> mutants.</p