Pinky Kain

Odour receptors and neurons for DEET and new insect repellents

There are major impediments to finding improved DEET alternatives because the receptors causing olfactory repellency are unknown, and new chemicals require exorbitant costs to determine safety for human use. Here we identify DEET-sensitive neurons in a pit-like structure in the Drosophila melanogaster antenna called the sacculus. They express a highly conserved receptor, Ir40a, and flies in which these neurons are silenced or Ir40a is knocked down lose avoidance to DEET. We used a computational structure–activity screen of >400,000 compounds that identified >100 natural compounds as candidate repellents. We tested several and found that most activate Ir40a+ neurons and are repellents for Drosophila. These compounds are also strong repellents for mosquitoes. The candidates contain chemicals that do not dissolve plastic, are affordable and smell mildly like grapes, with three considered safe in human foods. Our findings pave the way to discover new generations of repellents that will help fight deadly insect-borne diseases worldwide.

Reduced Odor Responses from Antennal Neurons of Gqα, Phospholipase Cβ, and rdgA Mutants in Drosophila Support a Role for a Phospholipid Intermediate in Insect Olfactory Transduction

Mechanisms by which G-protein-coupled odorant receptors transduce information in insects still need elucidation. We show that mutations in the Drosophila gene for Gqα (dgq) significantly reduce both the amplitude of the field potentials recorded from the whole antenna in responses to odorants as well as the frequency of evoked responses of individual sensory neurons. This requirement for Gqα is for adult function and not during antennal development. Conversely, brief expression of a dominant-active form of Gqα in adults leads to enhanced odor responses. To understand signaling downstream of Gqα in olfactory sensory neurons, genetic interactions of dgq were tested with mutants in genes known to affect phospholipid signaling. dgq mutant phenotypes were further enhanced by mutants in a PLCβ (phospholipase Cβ) gene, plc21C. Interestingly although, the olfactory phenotype of mutant alleles of diacylglycerol kinase (rdgA) was rescued by dgq mutant alleles. Our results suggest that Gqα-mediated olfactory transduction in Drosophila requires a phospholipid second messenger the levels of which are regulated by a cycle of phosphatidylinositol 1,4-bisphosphate breakdown and regeneration.

Secondary taste neurons that convey sweet taste and starvation in the Drosophila brain.

The gustatory system provides vital sensory information to determine feeding and appetitive learning behaviors. Very little is known, however, about higher-order gustatory circuits in the highly tractable model for neurobiology, Drosophila melanogaster. Here we report second-order sweet gustatory projection neurons (sGPNs) in the Drosophila brain using a powerful behavioral screen. Silencing neuronal activity reduces appetitive behaviors, whereas inducible activation results in food acceptance via proboscis extensions. sGPNs show functional connectivity with Gr5a(+) sweet taste neurons and are activated upon sucrose application to the labellum. By tracing sGPN axons, we identify the antennal mechanosensory and motor center (AMMC) as an immediate higher-order processing center for sweet taste. Interestingly, starvation increases sucrose sensitivity of the sGPNs in the AMMC, suggesting that hunger modulates the responsiveness of the secondary sweet taste relay. Together, our results provide a foundation for studying gustatory processing and its modulation by the internal nutrient state.

Kinesin Heavy Chain Function in Drosophila Glial Cells Controls Neuronal Activity

Kinesin heavy chain (Khc) is crucially required for axonal transport and khc mutants show axonal swellings and paralysis. Here, we demonstrate that in Drosophila khc is equally important in glial cells. Glial-specific downregulation of khc by RNA interference suppresses neuronal excitability and results in spastic flies. The specificity of the phenotype was verified by interspecies rescue experiments and further mutant analyses. Khc is mostly required in the subperineurial glia forming the blood–brain barrier. Following glial-specific knockdown, peripheral nerves are swollen with maldistributed mitochondria. To better understand khc function, we determined Khc-dependent Rab proteins in glia and present evidence that Neurexin IV, a well known blood–brain barrier constituent, is one of the relevant cargo proteins. Our work shows that the role of Khc for neuronal excitability must be considered in the light of its necessity for directed transport in glia.

Mutants in Phospholipid Signaling Attenuate the Behavioral Response of Adult Drosophila to Trehalose

In Drosophila melanogaster, gustatory receptor genes (Grs) encode putative G-protein-coupled receptors (GPCRs) that are expressed in gustatory receptor neurons (GRNs). One of the Gr genes, Gr5a, encodes a receptor for trehalose that is expressed in a subset of GRNs. Although a role for the G protein, Gsα, has been shown in Gr5a-expressing taste neurons, there is the residual responses to trehalose in Gsα mutants which could suggest additional transduction mechanisms. Expression and genetic analysis of the heterotrimeric G-protein subunit, Gq, shown here suggest involvement of this Gα subunit in trehalose perception in Drosophila. A green fluorescent protein reporter of Gq expression is detected in gustatory neurons in the labellum, tarsal segments, and wing margins. Animals heterozygous for dgq mutations and RNA interference-mediated knockdown of dgq showed reduced responses to trehalose in the proboscis extension reflex assay and feeding behavior assay. These defects were rescued by targeted expression of the wild-type dgqα transgene in the GRNs. These data together with observations from other mutants in phospholipid signaling provide insights into the mechanisms of taste transduction in Drosophila.

Drosophila mutants in phospholipid signaling have reduced olfactory responses as adults and larvae.

In this paper, we show that mutants in the gene stambhA (stmA), which encodes a putative phosphatidylinositol 4,5 bisphosphate-diacylglycerol lipase, exhibit a significant reduction in the amplitudes of odor-evoked responses recorded from the antennal surface of adult Drosophila. This lends support to previously published findings that olfactory transduction in Drosophila requires a phospholipid intermediate. Mutations in stmA also affect the olfactory behavior response of larvae. Moreover, there is a requirement for Gqα and phospholipase Cβ function in larval olfaction. The results suggest that larval olfactory transduction, like that of the adult, utilizes a phospholipid second messenger, generated by the activation of Gqα and Plcβ21c, and modulated by the stmA gene product.

Repellent compound with larger protective zone than DEET identified through activity-screening of Ir40a neurons, does not require Or function

The widely used insect repellent DEET has a limited spatial zone of protection, requiring it to be applied over all exposed areas of skin. Identification of insect DEET-sensing neurons expressing a highly conserved Ionotropic receptor, Ir40a, provides an opportunity to identify new structural classes of volatile agonists as potential spatial repellents. By imaging the activity of the Ir40a+ neurons in D. melanogaster expressing the calcium sensitive GCaMP3 protein, we identify a strong agonist, 4-methylpiperidine, with a much higher vapor pressure than DEET. Behavioral testing reveals that 4-methylpiperidine repels Aedes aegypti, which is consistent with our model that Ir40a marks a conserved innate aversive pathway. Using a spatial repellency assay we demonstrate that 4-methylpiperidine applied to one part of the hand repels mosquitoes on another part effectively, whereas DEET cannot do so. Using orco mutant A. aegypti we demonstrate that avoidance to 4- methypiperidine is not dependent on Or family function. Additional testing of orco mutant mosquitoes demonstrates that they are also effectively repelled by DEET, without coming in contact with it, in heat attraction assays. Together, these results support our initial observations that the conserved Ir pathway plays a key role in olfactory repellency and can be used to identify new classes of repellents.

Mutants in Drosophila TRPC Channels Reduce Olfactory Sensitivity to Carbon Dioxide

Background Members of the canonical Transient Receptor Potential (TRPC) class of cationic channels function downstream of Gαq and PLCβ in Drosophila photoreceptors for transducing visual stimuli. Gαq has recently been implicated in olfactory sensing of carbon dioxide (CO2) and other odorants. Here we investigated the role of PLCβ and TRPC channels for sensing CO2 in Drosophila. Methodology/Principal Findings Through behavioral assays it was demonstrated that Drosophila mutants for plc21c, trp and trpl have a reduced sensitivity for CO2. Immuno-histochemical staining for TRP, TRPL and TRPγ indicates that all three channels are expressed in Drosophila antennae including the sensory neurons that express CO2 receptors. Electrophysiological recordings obtained from the antennae of protein null alleles of TRP (trp343) and TRPL (trpl302), showed that the sensory response to multiple concentrations of CO2 was reduced. However, trpl302; trp343 double mutants still have a residual response to CO2. Down-regulation of TRPC channels specifically in CO2 sensing olfactory neurons reduced the response to CO2 and this reduction was obtained even upon down-regulation of the TRPCs in adult olfactory sensory neurons. Thus the reduced response to CO2 obtained from the antennae of TRPC RNAi strains is not due to a developmental defect. Conclusion These observations show that reduction in TRPC channel function significantly reduces the sensitivity of the olfactory response to CO2 concentrations of 5% or less in adult Drosophila. It is possible that the CO2 receptors Gr63a and Gr21a activate the TRPC channels through Gαq and PLC21C.

Retraction: Odour receptors and neurons for DEET and new insect repellents

This corrects the article DOI: 10.1038/nature12594