Summary Animals are often confronted with the decision as to consume a diet that contains competing attractive and aversive compounds. Here, using the fruit fly, Drosophila melanogaster, we describe a mechanism that influences this decision. Addition of bitter compounds to sucrose suppressed feeding behavior, and this inhibition depended on the odorant binding protein, OBP49a. In wild-type flies, bitter compounds suppressed sucrose-induced action potentials, and the inhibition was impaired in Obp49a mutants. However, loss of OBP49a did not affect action potentials in sugar- or bitter-activated gustatory receptor neurons (GRNs) when the GRNs were presented with just one type of tastant. OBP49a was expressed in accessory cells, and acted non-cell autonomously to attenuate nerve firings in sugar-activated GRNs when bitter compounds were combined with sucrose. These findings demonstrate an unexpected role for an OBP in taste, and identify a molecular player involved in the integration of opposing attractive and aversive gustatory inputs.

The activation of several transcription factors is required for the elimination of infectious pathogens via the innate immune response. The transcription factors NF-κB, AP-1, and STAT play major roles in the synthesis of immune effector molecules during innate immune responses. However, the fact that these immune responses can have cytotoxic effects requires their tight regulation to achieve restricted and transient activation, and mis-regulation of the damping process has pathological consequences. Here we show that AP-1 and STAT are themselves the major inhibitors responsible for damping NF-κB–mediated transcriptional activation during the innate immune response in Drosophila. As the levels of dAP-1 and Stat92E increase due to continuous immune signaling, they play a repressive role by forming a repressosome complex with the Drosophila HMG protein, Dsp1. The dAP-1–, Stat92E-, and Dsp1-containing complexes replace Relish at the promoters of diverse immune effector genes by binding to evolutionarily conserved cis-elements, and they recruit histone deacetylase to inhibit transcription. Reduction by mutation of dAP-1, Stat92E, or Dsp1 results in hyperactivation of Relish target genes and reduces the viability of bacterially infected flies despite more efficient pathogen clearance. These defects are rescued by reducing the Relish copy number, thus confirming that mis-regulation of Relish, not inadequate activation of dAP-1, Stat92E, or Dsp1 target genes, is responsible for the reduced survival of the mutants. We conclude that an inhibitory effect of AP-1 and STAT on NF-κB is required for properly balanced immune responses and appears to be evolutionarily conserved.

Insect survival depends on contact chemosensation to sense and avoid consuming plant-derived insecticides, such as l-canavanine. Members of a family of ~60 gustatory receptors (GRs) comprise the main peripheral receptors responsible for taste sensation in Drosophila. However, the roles of most Drosophila GRs are unknown. In addition to GRs, a G-protein coupled receptor (GPCR), DmXR, has been reported to be required for detecting l-canavanine. Here, we showed that GRs are essential for responding to l-canavanine, and that flies missing DmXR displayed normal l-canavanine avoidance and l-canavanine evoked action potentials. Mutations disrupting either Gr8a or Gr66a resulted in an inability to detect l-canavanine. We found that l-canavanine stimulated action potentials in S-type sensilla, which are where Gr8a and Gr66a were both expressed, but not in Gr66a-expressing sensilla that did not express Gr8a. l-canavanine-induced action potentials were also abolished in the Gr8a and Gr66a mutant animals. Gr8a was narrowly required for responding to l-canavanine, in contrast to Gr66a, which was broadly required for responding to other noxious tastants. Our data suggest that GR8a and GR66a are subunits of an l-canavanine receptor, and that GR8a contributes to the specificity for l-canavanine.

Animals discriminate nutritious food from toxic substances using their sense of taste. Since taste perception requires taste receptor cells to come into contact with water-soluble chemicals, it is a form of contact chemosensation. Concurrent with that contact, mechanosensitive cells detect the texture of food and also contribute to the regulation of feeding. Little is known, however, about the extent to which chemosensitive and mechanosensitive circuits interact. Here, we show Drosophila prefers soft food at the expense of sweetness and that this preference requires labellar mechanosensory neurons (MNs) and the mechanosensory channel Nanchung. Activation of these labellar MNs causes GABAergic inhibition of sweet-sensing gustatory receptor neurons, reducing the perceived intensity of a sweet stimulus. These findings expand our understanding of the ways different sensory modalities cooperate to shape animal behaviour.

The ability to detect toxic compounds in foods is essential for animal survival. However, the minimal subunit composition of gustatory receptors required for sensing aversive chemicals in Drosophila is unknown. Here we report that three gustatory receptors, GR8a, GR66a and GR98b function together in the detection of L-canavanine, a plant-derived insecticide. Ectopic co-expression of Gr8a and Gr98b in Gr66a-expressing, bitter-sensing gustatory receptor neurons (GRNs) confers responsiveness to L-canavanine. Furthermore, misexpression of all three Grs enables salt- or sweet-sensing GRNs to respond to L-canavanine. Introduction of these Grs in sweet-sensing GRNs switches L-canavanine from an aversive to an attractive compound. Co-expression of GR8a, GR66a and GR98b in Drosophila S2 cells induces an L-canavanine-activated nonselective cation conductance. We conclude that three GRs collaborate to produce a functional L-canavanine receptor. Thus, our results clarify the full set of GRs underlying the detection of a toxic tastant that drives avoidance behaviour in an insect.

Phagocytosis of invading microbes requires dynamic rearrangement of the plasma membrane and its associated cytoskeletal actin network. The polarization of Cdc42 and Rac1 Rho GTPases to the site of plasma membrane protrusion is responsible for the remodeling of actin structures. However, the mechanism of Rho GTPase recruitment to these sites and the identities of accessory molecules involved in this process are not well understood. In this study, we uncovered several new components involved in innate immunity in Drosophila melanogaster. Our data demonstrate that Rab35 is a regulator of vesicle transport required specifically for phagocytosis. Moreover, recruitment of Cdc42 and Rac1 to the sites of filopodium and lamellipodium formation is Rab35 dependent and occurs by way of microtubule tracks. These results implicate Rab35 as the immune cell-specific regulator of vesicle transport within the actin-remodeling complex.

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-κB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila.

Mechanically gated ion channels convert sound into an electrical signal for the sense of hearing. In Drosophila melanogaster, several transient receptor potential (TRP) channels have been implicated to be involved in this process. TRPN (NompC) and TRPV (Inactive) channels are localized in the distal and proximal ciliary zones of auditory receptor neurons, respectively. This segregated ciliary localization suggests distinct roles in auditory transduction. However, the regulation of this localization is not fully understood. Here we show that the Drosophila Tubby homolog, King tubby (hereafter called dTULP) regulates ciliary localization of TRPs. dTULP-deficient flies show uncoordinated movement and complete loss of sound-evoked action potentials. Inactive and NompC are mislocalized in the cilia of auditory receptor neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and suggests that dTULP is a protein that regulates ciliary neurosensory functions.