Chemogenetic activation of mammalian brain neurons expressing insect Ionotropic Receptors by systemic ligand precursor administration.

Chemogenetic approaches employing ligand-gated ion channels are advantageous regarding manipulation of target neuronal population functions independently of endogenous second messenger pathways. Among them, Ionotropic Receptor (IR)-mediated neuronal activation (IRNA) allows stimulation of mammalian neurons that heterologously express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the original protocol, phenylacetic acid, a ligand of the IR84a/IR8a complex, was locally injected into a brain region due to its low permeability of the blood-brain barrier. To circumvent this invasive injection, we sought to develop a strategy of peripheral administration with a precursor of phenylacetic acid, phenylacetic acid methyl ester, which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. This strategy was validated by electrophysiological, biochemical, brain-imaging, and behavioral analyses, demonstrating high utility of systemic IRNA technology in the remote activation of target neurons in the brain.

Sensory neuron population expansion enhances odor tracking without sensitizing projection neurons.

The evolutionary expansion of sensory neuron populations detecting important environmental cues is widespread, but functionally enigmatic. We investigated this phenomenon through comparison of homologous neural pathways of Drosophila melanogaster and its close relative Drosophila sechellia , an extreme specialist for Morinda citrifolia noni fruit. D. sechellia has evolved species-specific expansions in select, noni-detecting olfactory sensory neuron (OSN) populations, through multigenic changes. Activation and inhibition of defined proportions of neurons demonstrate that OSN population increases contribute to stronger, more persistent, noni-odor tracking behavior. These sensory neuron expansions result in increased synaptic connections with their projection neuron (PN) partners, which are conserved in number between species. Surprisingly, having more OSNs does not lead to greater odor-evoked PN sensitivity or reliability. Rather, pathways with increased sensory pooling exhibit reduced PN adaptation, likely through weakened lateral inhibition. Our work reveals an unexpected functional impact of sensory neuron expansions to explain ecologically-relevant, species-specific behavior.

Remote homolog detection places insect chemoreceptors in a cryptic protein superfamily spanning the tree of life.

Many proteins exist in the so-called "twilight zone" of sequence alignment, where low pairwise sequence identity makes it difficult to determine homology and phylogeny.1,2 As protein tertiary structure is often more conserved,3 recent advances in ab initio protein folding have made structure-based identification of putative homologs feasible.4,5,6 We present a pipeline for the identification and characterization of distant homologs and apply it to 7-transmembrane-domain ion channels (7TMICs), a protein group founded by insect odorant and gustatory receptors. Previous sequence and limited structure-based searches identified putatively related proteins, mainly in other animals and plants.7,8,9,10 However, very few 7TMICs have been identified in non-animal, non-plant taxa. Moreover, these proteins' remarkable sequence dissimilarity made it uncertain whether disparate 7TMIC types (Gr/Or, Grl, GRL, DUF3537, PHTF, and GrlHz) are homologous or convergent, leaving their evolutionary history unresolved. Our pipeline identified thousands of new 7TMICs in archaea, bacteria, and unicellular eukaryotes. Using graph-based analyses and protein language models to extract family-wide signatures, we demonstrate that 7TMICs have structure and sequence similarity, supporting homology. Through sequence- and structure-based phylogenetics, we classify eukaryotic 7TMICs into two families (Class-A and Class-B), which are the result of a gene duplication predating the split(s) leading to Amorphea (animals, fungi, and allies) and Diaphoretickes (plants and allies). Our work reveals 7TMICs as a cryptic superfamily, with origins close to the evolution of cellular life. More generally, this study serves as a methodological proof of principle for the identification of extremely distant protein homologs.

Odor-regulated oviposition behavior in an ecological specialist.

Colonization of a novel ecological niche can require, or be driven by, evolution of an animal's behaviors promoting their reproductive success. We investigated the evolution and sensory basis of oviposition in Drosophila sechellia, a close relative of Drosophila melanogaster that exhibits extreme specialism for Morinda citrifolia noni fruit. D. sechellia produces fewer eggs than other drosophilids and lays these almost exclusively on noni substrates. We show that visual, textural and social cues do not explain this species-specific preference. By contrast, we find that loss of olfactory input in D. sechellia, but not D. melanogaster, essentially abolishes egg-laying, suggesting that olfaction gates gustatory-driven noni preference. Noni odors are detected by redundant olfactory pathways, but we discover a role for hexanoic acid and the cognate Ionotropic receptor 75b (Ir75b) in odor-evoked oviposition. Through receptor exchange in D. melanogaster, we provide evidence for a causal contribution of odor-tuning changes in Ir75b to the evolution of D. sechellia's oviposition behavior.

Evolution of connectivity architecture in the Drosophila mushroom body.

Brain evolution has primarily been studied at the macroscopic level by comparing the relative size of homologous brain centers between species. How neuronal circuits change at the cellular level over evolutionary time remains largely unanswered. Here, using a phylogenetically informed framework, we compare the olfactory circuits of three closely related Drosophila species that differ radically in their chemical ecology: the generalists Drosophila melanogaster and Drosophila simulans that feed on fermenting fruit, and Drosophila sechellia that specializes on ripe noni fruit. We examine a central part of the olfactory circuit that has not yet been investigated in these species - the connections between the projection neurons of the antennal lobe and the Kenyon cells of the mushroom body, an associative brain center - to identify species-specific connectivity patterns. We found that neurons encoding food odors - the DC3 neurons in D. melanogaster and D. simulans and the DL2d neurons in D. sechellia - connect more frequently with Kenyon cells, giving rise to species-specific biases in connectivity. These species-specific differences in connectivity reflect two distinct neuronal phenotypes: in the number of projection neurons or in the number of presynaptic boutons formed by individual projection neurons. Finally, behavioral analyses suggest that such increased connectivity enhances learning performance in an associative task. Our study shows how fine-grained aspects of connectivity architecture in an associative brain center can change during evolution to reflect the chemical ecology of a species.

Structural screens identify candidate human homologs of insect chemoreceptors and cryptic Drosophila gustatory receptor-like proteins.

Insect odorant receptors and gustatory receptors define a superfamily of seven transmembrane domain ion channels (referred to here as 7TMICs), with homologs identified across Animalia except Chordata. Previously, we used sequence-based screening methods to reveal conservation of this family in unicellular eukaryotes and plants (DUF3537 proteins) (Benton et al., 2020). Here, we combine three-dimensional structure-based screening, ab initio protein folding predictions, phylogenetics, and expression analyses to characterize additional candidate homologs with tertiary but little or no primary structural similarity to known 7TMICs, including proteins in disease-causing Trypanosoma. Unexpectedly, we identify structural similarity between 7TMICs and PHTF proteins, a deeply conserved family of unknown function, whose human orthologs display enriched expression in testis, cerebellum, and muscle. We also discover divergent groups of 7TMICs in insects, which we term the gustatory receptor-like (Grl) proteins. Several Drosophila melanogaster Grls display selective expression in subsets of taste neurons, suggesting that they are previously unrecognized insect chemoreceptors. Although we cannot exclude the possibility of remarkable structural convergence, our findings support the origin of 7TMICs in a eukaryotic common ancestor, counter previous assumptions of complete loss of 7TMICs in Chordata, and highlight the extreme evolvability of this protein fold, which likely underlies its functional diversification in different cellular contexts.

Chemosensory Coding in Drosophila Single Sensilla.

The chemical senses-smell and taste-detect and discriminate an enormous diversity of environmental stimuli and provide fascinating but challenging models to investigate how sensory cues are represented in the brain. Important stimulus-coding events occur in peripheral sensory neurons, which express specific combinations of chemosensory receptors with defined ligand-response profiles. These receptors convert ligand recognition into spatial and temporal patterns of neural activity that are transmitted to, and interpreted in, central brain regions. Drosophila melanogaster provides an attractive model to study chemosensory coding because it possesses relatively simple peripheral olfactory and gustatory systems that display many organizational parallels to those of vertebrates. Moreover, nearly all peripheral chemosensory neurons have been molecularly characterized and are accessible for physiological analysis, as they are exposed on the surface of sensory organs housed in specialized hairs called sensilla. Here, we briefly review anatomical, molecular, and physiological properties of adult Drosophila olfactory and gustatory systems and provide background to methods for electrophysiological recordings of ligand-evoked activity from different types of chemosensory sensilla.

Recording from Fly Olfactory Sensilla.

Olfactory systems detect and discriminate an enormous diversity of volatile environmental stimuli and provide important paradigms to investigate how sensory cues are represented in the brain. Key stimulus-coding events occur in peripheral olfactory sensory neurons, which typically express a single olfactory receptor-from a large repertoire encoded in the genome-with a defined ligand-response profile. These receptors convert odor ligand recognition into spatial and temporal patterns of neural activity that are transmitted to, and interpreted in, central brain regions. Drosophila provides an attractive model to study olfactory coding because it possesses a relatively simple peripheral olfactory system that displays many organizational parallels to those of vertebrates. Moreover, nearly all olfactory sensory neurons have been molecularly characterized and are accessible for physiological analysis, as they are exposed on the surface of sensory organs (antennae and maxillary palps) housed in specialized hairs called sensilla. This protocol describes how to perform recordings of odor-evoked activity from Drosophila olfactory sensilla, covering the basics of sample preparation, setting up the electrophysiology rig, assembling an odor stimulus-delivery device, and data analysis. The methodology can be used to characterize the ligand-recognition properties of most olfactory sensory neurons and the role of olfactory receptors (and other molecular components) in signal transduction.

Recording from Fly Taste Sensilla.

Gustatory systems sense chemicals upon contact and provide a model to investigate how these stimuli are encoded to inform various behavioral decisions including choice of foods, egg-laying sites, and mating partners. Multiple organs in the body house peripheral gustatory sensory neurons, the axons of which project to discrete regions in the subesophageal zone and ventral ganglion, representing both the location and quality of the taste stimulus. Taste neurons are broadly divided into subpopulations associated with either positive or negative behavioral valence, each expressing combinations of taste receptors-in some cases, more than 30 receptors-encoded by one or more chemosensory gene families that together determine their chemical response properties. Drosophila provides a powerful model to study gustatory coding because a majority of the taste sensory units (sensilla) are present in external taste organs (labellum and legs) and are accessible for electrophysiological analysis of tastant-evoked responses. Moreover, a large body of work on the basic characteristics of individual taste neurons housed in a sensillum, as well as on functional surveys of entire taste organs, provides a foundation for investigating further questions about taste coding, adaptability, and evolution. This protocol describes how to perform recordings of stimulus-evoked activity from Drosophila taste sensilla covering the basics of setting up the electrophysiology rig and stimulus-delivery device, sample preparation, and performing and analyzing the recordings.

Drosophila olfaction: past, present and future.

Among the many wonders of nature, the sense of smell of the fly Drosophila melanogaster might seem, at first glance, of esoteric interest. Nevertheless, for over a century, the 'nose' of this insect has been an extraordinary system to explore questions in animal behaviour, ecology and evolution, neuroscience, physiology and molecular genetics. The insights gained are relevant for our understanding of the sensory biology of vertebrates, including humans, and other insect species, encompassing those detrimental to human health. Here, I present an overview of our current knowledge of D. melanogaster olfaction, from molecules to behaviours, with an emphasis on the historical motivations of studies and illustration of how technical innovations have enabled advances. I also highlight some of the pressing and long-term questions.

Pheromone sensing in Drosophila requires support cell-expressed Osiris 8.

BACKGROUND: The nose of most animals comprises multiple sensory subsystems, which are defined by the expression of different olfactory receptor families. Drosophila melanogaster antennae contain two morphologically and functionally distinct subsystems that express odorant receptors (Ors) or ionotropic receptors (Irs). Although these receptors have been thoroughly characterized in this species, the subsystem-specific expression and roles of other genes are much less well-understood. RESULTS: Here we generate subsystem-specific transcriptomic datasets to identify hundreds of genes, encoding diverse protein classes, that are selectively enriched in either Or or Ir subsystems. Using single-cell antennal transcriptomic data and RNA in situ hybridization, we find that most neuronal genes-other than sensory receptor genes-are broadly expressed within the subsystems. By contrast, we identify many non-neuronal genes that exhibit highly selective expression, revealing substantial molecular heterogeneity in the non-neuronal cellular components of the olfactory subsystems. We characterize one Or subsystem-specific non-neuronal molecule, Osiris 8 (Osi8), a conserved member of a large, insect-specific family of transmembrane proteins. Osi8 is expressed in the membranes of tormogen support cells of pheromone-sensing trichoid sensilla. Loss of Osi8 does not have obvious impact on trichoid sensillar development or basal neuronal activity, but abolishes high sensitivity responses to pheromone ligands. CONCLUSIONS: This work identifies a new protein required for insect pheromone detection, emphasizes the importance of support cells in neuronal sensory functions, and provides a resource for future characterization of other olfactory subsystem-specific genes.

Sweet sensors support stressed cell survival.

Gustatory receptors (Grs) are well known for their functions in sensory neurons in detecting food and toxins. An intriguing new study in PLOS Biology provides evidence for a role for Grs in Drosophila epithelia in protecting stressed cells from proteotoxicity.

Copy number changes in co-expressed odorant receptor genes enable selection for sensory differences in drosophilid species.

Despite numerous examples of chemoreceptor gene family expansions and contractions, how these relate to modifications in the sensory neuron populations in which they are expressed remains unclear. Drosophila melanogaster's odorant receptor (Or) family is ideal for addressing this question because most Ors are expressed in distinct olfactory sensory neuron (OSN) types. Between-species changes in Or copy number may therefore indicate increases or reductions in the number of OSN populations. Here we investigated the Or67a subfamily, which exhibits copy number variation in D. melanogaster and its closest relatives: D. simulans, D. sechellia and D. mauritiana. These species' common ancestor had three Or67a paralogues that had already diverged adaptively. Following speciation, two Or67a paralogues were lost independently in D. melanogaster and D. sechellia, with ongoing positive selection shaping the intact genes. Unexpectedly, the functionally diverged Or67a paralogues in D. simulans are co-expressed in a single neuron population, which projects to a glomerulus homologous to that innervated by Or67a neurons in D. melanogaster. Thus, while sensory pathway neuroanatomy is conserved, independent selection on co-expressed receptors has contributed to species-specific peripheral coding. This work reveals a type of adaptive change largely overlooked for olfactory evolution, raising the possibility that similar processes influence other cases of insect Or co-expression.

Firm partisan positioning, polarization, and risk communication: Examining voluntary disclosures on COVID-19.

Research Summary: The COVID-19 pandemic will rank among the greatest challenges many executives will have faced and not only due to the operational challenges it posed. Upon entering the U.S. context, the disease was immediately politically polarized, with clear partisan splits forming in risk perceptions of the disease unrelated to science. We exploit this context to examine whether firms' partisan positioning affects whether and how they communicate risk to their investors on a polarized public policy issue. To do so, we examine the covariation between firms' disclosure of COVID-19 risks and the partisanship of their political giving. Our analysis of earnings call and campaign contribution data for the S&P 500 reveals a positive association between a firm's contributions to Democrats and its disclosure of COVID-19 risks. Managerial Summary: From its onset in the United States, attitudes toward and discourse around the COVID-19 pandemic was heavily politicized and perceptions of the disease's risks were seen as more serious by Democratic-identifying individuals than Republican identifiers. In this study, we examine whether this pattern also holds for U.S. publicly traded firms, who can also stake out a political position through their corporate political action committee campaign contributions. In analyses of earnings call transcripts from the first quarter of 2020, we show that the more Republican-leaning (Democrat-leaning) a firm's campaign contributions are, the less (more) likely it was to voluntarily disclose risks related to COVID-19. We argue that these findings hold implications for parties interested in interpreting firm's risk disclosures on politically polarized issues.

Gender inequality in relational position-taking: An analysis of intra-organizational job mobility networks.

We conceptualize within-organization job mobility as a position-taking process, arguing that the structure and outcome of claims over positions are characteristics of organizational inequality regimes. Drawing on data from 10 distribution centers from a large U.S. firm, we examine gendered job mobility as the observed network of workers moving among jobs. Results from network analysis and meta-regression reveal that in the firm examined, workers tend to move between jobs with similar gender compositions, that mobility lattices tend to be more ladder-like for male-concentrated jobs but more circuitous for female-concentrated jobs, and that there is less upward mobility overall in organizations with higher levels of wage inequality. Both organization level inequalities and the relationship between positions within organizations condition mobility. While we do not observe discursive claims on positions, we argue that these are the underlying mechanisms driving gendered job mobility.

Olfactory Receptor Gene Regulation in Insects: Multiple Mechanisms for Singular Expression.

The singular expression of insect olfactory receptors in specific populations of olfactory sensory neurons is fundamental to the encoding of odors in patterns of neuronal activity in the brain. How a receptor gene is selected, from among a large repertoire in the genome, to be expressed in a particular neuron is an outstanding question. Focusing on Drosophila melanogaster, where most investigations have been performed, but incorporating recent insights from other insect species, we review the multilevel regulatory mechanisms of olfactory receptor expression. We discuss how cis-regulatory elements, trans-acting factors, chromatin modifications, and feedback pathways collaborate to activate and maintain expression of the chosen receptor (and to suppress others), highlighting similarities and differences with the mechanisms underlying singular receptor expression in mammals. We also consider the plasticity of receptor regulation in response to environmental cues and internal state during the lifetime of an individual, as well as the evolution of novel expression patterns over longer timescales. Finally, we describe the mechanisms and potential significance of examples of receptor co-expression.

Molecular reconstruction of recurrent evolutionary switching in olfactory receptor specificity.

Olfactory receptor repertoires exhibit remarkable functional diversity, but how these proteins have evolved is poorly understood. Through analysis of extant and ancestrally reconstructed drosophilid olfactory receptors from the Ionotropic receptor (Ir) family, we investigated evolution of two organic acid-sensing receptors, Ir75a and Ir75b. Despite their low amino acid identity, we identify a common 'hotspot' in their ligand-binding pocket that has a major effect on changing the specificity of both Irs, as well as at least two distinct functional transitions in Ir75a during evolution. Moreover, we show that odor specificity is refined by changes in additional, receptor-specific sites, including those outside the ligand-binding pocket. Our work reveals how a core, common determinant of ligand-tuning acts within epistatic and allosteric networks of substitutions to lead to functional evolution of olfactory receptors.

Drosophila sechellia: A Genetic Model for Behavioral Evolution and Neuroecology.

Defining the mechanisms by which animals adapt to their ecological niche is an important problem bridging evolution, genetics, and neurobiology. We review the establishment of a powerful genetic model for comparative behavioral analysis and neuroecology, Drosophila sechellia. This island-endemic fly species is closely related to several cosmopolitan generalists, including Drosophila melanogaster, but has evolved extreme specialism, feeding and reproducing exclusively on the noni fruit of the tropical shrub Morinda citrifolia. We first describe the development and use of genetic approaches to facilitate genotype/phenotype associations in these drosophilids. Next, we survey the behavioral, physiological, and morphological adaptations of D. sechellia throughout its life cycle and outline our current understanding of the genetic and cellular basis of these traits. Finally, we discuss the principles this knowledge begins to establish in the context of host specialization, speciation, and the neurobiology of behavioral evolution and consider open questions and challenges in the field.

Olfactory receptor-dependent receptor repression in Drosophila.

In olfactory systems across phyla, most sensory neurons express a single olfactory receptor gene selected from a large genomic repertoire. We describe previously unknown receptor gene-dependent mechanisms that ensure singular expression of receptors encoded by a tandem gene array [Ionotropic receptor 75c (Ir75c), Ir75b, and Ir75a, organized 5' to 3'] in Drosophila melanogaster Transcription from upstream genes in the cluster runs through the coding region of downstream loci and inhibits their expression in cis, most likely via transcriptional interference. Moreover, Ir75c blocks accumulation of other receptor proteins in trans through a protein-dependent, posttranscriptional mechanism. These repression mechanisms operate in endogenous neurons, in conjunction with cell type-specific gene regulatory networks, to ensure unique receptor expression. Our data provide evidence for inter-olfactory receptor regulation in invertebrates and highlight unprecedented, but potentially widespread, mechanisms for ensuring exclusive expression of chemosensory receptors, and other protein families, encoded by tandemly arranged genes.

Targeted molecular profiling of rare olfactory sensory neurons identifies fate, wiring, and functional determinants.

Determining the molecular properties of neurons is essential to understand their development, function and evolution. Using Targeted DamID (TaDa), we characterize RNA polymerase II occupancy and chromatin accessibility in selected Ionotropic receptor (Ir)-expressing olfactory sensory neurons in Drosophila. Although individual populations represent a minute fraction of cells, TaDa is sufficiently sensitive and specific to identify the expected receptor genes. Unique Ir expression is not consistently associated with differences in chromatin accessibility, but rather to distinct transcription factor profiles. Genes that are heterogeneously expressed across populations are enriched for neurodevelopmental factors, and we identify functions for the POU-domain protein Pdm3 as a genetic switch of Ir neuron fate, and the atypical cadherin Flamingo in segregation of neurons into discrete glomeruli. Together this study reveals the effectiveness of TaDa in profiling rare neural populations, identifies new roles for a transcription factor and a neuronal guidance molecule, and provides valuable datasets for future exploration.