Biology subjects

Benton, R.

Publications and source records attributed to Benton, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Temporal niche partitioning through olfactory cell type evolution

Temporal niche partitioning enables species to use the same resource at different times, but the underlying mechanisms are unknown. We show that Drosophila sechellia, a specialist on Morinda citrifolia noni, exhibits narrow temporal preference for the most toxic, ripe fruit, reducing exposure to competition, parasitization and microbial infection that can occur on other stages. Chemical analysis highlighted ketones as signature odors of ripe noni. Comparative single-cell transcriptomic atlases revealed that D. sechellia has co-opted expression of a larval ketone receptor, Or45a, in an adult olfactory neuron population. The novel expression of Or45a can be ascribed solely to cis-regulatory changes and is sufficient to confer physiological sensitivity to ripe noni. Importantly, Or45a is required for D. sechellia's ripe noni preference, acting redundantly with a second ketone receptor, Or85c/b, whose neuron population has expanded in D. sechellia. Our results provide an unprecedented link between cell type evolution and ecologically-advantageous, temporal niche partitioning.

neuroscience↗

bioRxiv2026

Laboratory and wild Drosophila sechellia have conserved niche specialization phenotypes

A major challenge to investigating the proximate causes of ecological adaptation is the difficulty of studying the phenotypes of organisms in their natural environments. By necessity, many studies seeking to determine the genetic and cellular basis of adaptation therefore investigate potentially adaptive phenotypes under laboratory conditions where organisms are more easily experimentally manipulated. For laboratory models, it remains unclear if organisms maintained long term under laboratory conditions are representative of relatives in their natural environment. In recent years, Drosophila sechellia, a specialist species endemic to the Seychelles, has emerged as a (neuro)genetic model for studying the molecular basis of ecological adaptation. A multitude of studies have investigated the genetic and cellular basis of various aspects of this species specialization in a laboratory setting. However, the vast majority of these studies use laboratory strains of D. sechellia that were collected many decades ago, and have been maintained under conditions very different from their natural niche. Thus, it remains unclear if and how these strains resemble their wild counterparts. Here, we compare the phenotypes of these laboratory strains with recently-collected wild D. sechellia to ask if laboratory strains display a loss or degradation of phenotypes potentially involved in their specialization resulting from their long-term laboratory maintenance. Across several behavioral and anatomical phenotypes, we find a high degree of similarity between wild-caught and laboratory-maintained strains. Our results suggest that studies of the molecular mechanisms underlying D. sechellias phenotypes associated with specialization are likely representative of the evolution of these flies in the wild.

evolutionary biology↗

bioRxiv2025

Genome-wide association studies identify new candidate genes and tissues underlying resistance to a natural toxin in drosophilids

Many insects can rapidly evolve resistance to artificial insecticides through changes in toxin target proteins. Over longer timescales, insects have also evolved resistance to naturally-occurring toxins to exploit new ecological niches, but much less is known about the mechanisms underlying such adaptations. A classic example is Drosophila sechellia, an extreme specialist for the ripe noni fruit of Morinda citrifolia, which is toxic for other insects - including the close relatives D. simulans and D. melanogaster - due to nonis high content of octanoic acid (OA). The mechanistic bases underlying susceptibility and resistance to OA of different species remain unclear. Here, we first show that the species-specific tolerance of OA is independent of these drosophilids distinct microbiomes, reinforcing the notion that this trait is genetically encoded. Screening large, genetically-diverse panels of D. melanogaster and D. simulans strains revealed broad variation in OA resistance, with some lines surviving as well as D. sechellia. Resistance to OA does not correlate with resistance of these lines to other insecticides, implying a distinct toxicity mode-of-action. Genome-wide association and transcriptome-to-phenotype analyses identified multiple genes linked to OA resistance. These genes have diverse expression patterns and functions, including proteins involved in epithelial septate junction formation, lipid transport and tracheal morphogenesis. Loss-of-function analysis in D. melanogaster confirmed that at least two of these - Bez, a CD36-family fatty acid transporter, and CG13003, a putative extracellular matrix component - positively contribute to OA resistance. Integration of our findings with those from previous complementary genetic approaches supports a model in which OA has no singular target, and that resistance to this toxin is defined by multigenic and multi-tissue defense mechanisms.

evolutionary biology↗

bioRxiv2025

A GPCR signaling pathway in insect odor detection

Odor detection differs fundamentally in vertebrates, which use G protein-coupled receptors (GPCRs)1,2, and insects, which employ ion channels3,4. Here, we report the first evidence for a GPCR defining tuning properties of insect olfactory sensory neurons. Single-cell transcriptomics of the Drosophila melanogaster antenna identified selective expression of the G{gamma}30A subunit in acid-sensing Ir64a-DC4 neurons5,6. G{gamma}30A is essential for broadening responses to long-chain acids, acting with Gs, G{beta}13F, adenylate cyclase Ac13E and the Cngl channel. We further discovered that Cirl, a latrophilin-family GPCR7, is broadly-transcribed in the antenna but the protein is localized only in Ir64a-DC4 sensory cilia, dependent upon G{gamma}30A, but not Ir64a. Importantly, loss of Cirl also narrows Ir64a-DC4 tuning properties. Homologous neurons in Drosophila sechellia naturally exhibit narrow acid tuning, despite functional conservation of Ir64a; these differences correlate instead with lower expression of metabotropic components. Our findings reveal unexpected roles for GPCR/metabotropic signaling in olfactory detection and divergence in insects.

neuroscience↗

bioRxiv2025

Intersecting experimental evolution and CRISPR screens to identify novel insecticide resistance loci

Understanding toxin resistance in insects is key to appreciate niche adaptations but remains challenging due to its often-polygenic basis. A well-known example is the specialized association of Drosophila sechellia with noni fruit (Morinda citrifolia), which is toxic to most other insects, including the closely-related Drosophila simulans and Drosophila melanogaster. Toxicity of noni is due to its high concentration of octanoic acid (OA), but the mechanisms that determine sensitivity or resistance to OA in different species remain poorly understood. Here, we experimentally-evolved D. simulans with increased OA resistance, identifying multiple loci under selection. Cross-referencing these with a genome-wide, OA-resistance CRISPR screen in a D. melanogaster cell line highlighted two proteins: Kraken, a putative detoxification enzyme expressed in digestive and renal tissues, and Alkbh7, a mitochondrial protein linked to fatty acid metabolism. Both genes show elevated expression in D. sechellia and OA-resistant D. simulans. In D. melanogaster, kraken mutants are more OA-sensitive, while Alkbh7 overexpression increased OA resistance. Importantly, mutation of these genes in D. sechellia reduced OA tolerance. Our identification of genes underlying OA resistance in laboratory and natural contexts demonstrates how complementary, cross-species selection approaches can provide insights into complex mechanisms of toxin susceptibility and adaptation; such methods could also have practical applications in the characterization of natural and artificial insecticides.

evolutionary biology↗

bioRxiv2025

An integrated anatomical, functional and evolutionary view of the Drosophila olfactory system

The Drosophila melanogaster olfactory system is one of the most intensively studied parts of the nervous system in any animal. Composed of [~]60 independent olfactory neuron classes, with several associated hygrosensory and thermosensory pathways, it has been subject to diverse types of experimental analyses. However, synthesizing the available data is limited by the incompleteness and inconsistent nomenclature found in the literature. In this work, we first "complete" the peripheral sensory map through the identification of a previously uncharacterized antennal sensory neuron population expressing Or46aB, and the definition of an exceptional "hybrid" olfactory neuron class comprising functional Or and Ir receptors. Second, we survey developmental, anatomical, connectomic, functional and evolutionary studies to generate an integrated dataset of these sensory neuron pathways - and associated visualizations - creating an unprecedented comprehensive resource. Third, we illustrate the utility of the dataset to reveal relationships between different organizational properties of this sensory system, and the new questions these stimulate. These examples emphasize the power of this resource to promote further understanding of the construction, function and evolution of these neural circuits.

neuroscience↗

bioRxiv2025

Multilayer regulation underlies the functional precision and evolvability of the olfactory system

Sensory neurons must be reproducibly specified to permit accurate neural representation of external signals but also able to change during evolution. We studied this paradox in the Drosophila olfactory system by establishing a single-cell transcriptomic atlas of all developing antennal sensory lineages, including latent neural populations that normally undergo programmed cell death (PCD). This atlas reveals that transcriptional control is robust, but imperfect, in defining selective sensory receptor expression. A second layer of precision is afforded by the intersection of expression of functionally-interacting receptor subunits. A third layer is defined by stereotyped PCD patterning, which masks promiscuous receptor expression in neurons fated to die and removes "empty" neurons lacking receptors. Like receptor choice, PCD is under lineage-specific transcriptional control; promiscuity in this regulation leads to previously-unappreciated heterogeneity in neuronal numbers. Thus functional precision in the mature olfactory system belies developmental noise that might facilitate the evolution of sensory pathways.

developmental biology↗

bioRxiv2024

Olfactory projection neuron rewiring in the brain of an ecological specialist

Animals behaviours can vary greatly between even closely-related species. While changes in the sensory periphery have frequently been linked to species-specific behaviours, very little is known about if and how individual cell types in the central brain evolve. Here, we develop a set of advanced genetic tools to compare homologous neurons in Drosophila sechellia - which specialises on a single fruit - and Drosophila melanogaster. Through systematic morphological analysis of olfactory projection neurons (PNs), we reveal that global anatomy of these second-order neurons is conserved. However, high-resolution, quantitative comparisons identify a striking case of convergent rewiring of PNs in two distinct olfactory pathways critical for D. sechellias host location. Calcium imaging and labelling of pre-synaptic sites in these evolved PNs demonstrate that novel functional connections with third-order partners are formed in D. sechellia. This work demonstrates that peripheral sensory evolution is accompanied by highly-selective wiring changes in the central brain to facilitate ecological specialisation, and paves the way for systematic comparison of other cell types throughout the nervous system.

neuroscience↗

bioRxiv2023

Comparative single-cell transcriptomic atlases reveal conserved and divergent features of drosophilid central brains

To explore how brains change upon species evolution, we generated single-cell transcriptomic atlases of the central brains of three closely-related but ecologically-distinct drosophilids: the generalists Drosophila melanogaster and Drosophila simulans, and the noni fruit specialist Drosophila sechellia. The global cellular composition of these species central brains is well-conserved, but we predicted a few cell types (perineurial glia, sNPF and Dh44 peptidergic neurons) with divergent frequencies. Gene expression analysis revealed that distinct cell types within the central brain evolve at different rates and patterns; notably, glial cell types exhibit the greatest divergence between species. Compared to D. melanogaster, the cellular composition and gene expression patterns of the central brain in D. sechellia display greater deviation than those of D. simulans - despite their similar phylogenetic distance from D. melanogaster - that the distinctive ecological specialization of D. sechellia is reflected in the structure and function of its brain. Expression changes in D. sechellia encompass metabolic and ecdysone signaling genes, suggestive of adaptations to its novel ecological demands. Additional single-cell transcriptomic analysis on D. sechellia revealed genes and cell types responsive to dietary supplement with noni, pointing to glia as sites for both physiological and genetic adaptation to novel conditions. Our atlases represent the first comparative analyses of "whole" central brains, and provide a comprehensive foundation for studying the evolvability of nervous systems in a well-defined phylogenetic and ecological framework.

evolutionary biology↗

bioRxiv2023

Sensory neuron population expansion enhances odour tracking through relaxed projection neuron adaptation

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.

neuroscience↗

bioRxiv2023

Chemogenetic activation of target neurons expressing insect Ionotropic Receptors in the mammalian central nervous system by systemic administration of ligand precursors

The IR-mediated neuronal activation (IRNA) technology allows stimulation of neurons in the brain that heterologously-express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the current protocol, a ligand against the complex consisting of IR84a and IR8a subunits, phenylacetic acid (PhAc), is locally injected into a brain region, because of a low efficiency of PhAc for the delivery into the brain across the blood-brain barrier. To circumvent this invasive injection, here we developed a strategy for activation of target neurons in the brain through peripheral administration with a precursor of PhAc, methyl ester of PhAc (PhAcM), which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. Peripheral administration of with PhAcM activated IR84a/IR8a-expressing neurons in the locus coeruleus of mice and increased the release of neurotransmitters in their nerve terminal regions. (S)-2-phenylpropionic acid ((S)-PhPr) was newly identified as a ligand for IR84a/IR8a, and peripheral administration with the methyl ester of PhPr with the S-configuration [(S)-PhPrM] caused similar effects on the target neurons. In addition, cell-type specific expression of IR84a/IR8a complex in the striatum of rats was unilaterally induced with a viral vector based on the Cre-loxP system. Peripheral administration with PhAcM or (S)-PhPrM stimulated the neurotransmitter release in the ipsilateral terminal regions of the vector-injected striatum, and PhAcM administration resulted in rotational behavior. Finally, we demonstrated that the metabolites of the peripherally administered-radiolabeled (S)-PhPrM accumulated in the IR84a/IR8a-expressing region in the striatum of the vector-injected rats. These results demonstrate that the systemic IRNA technique provides a powerful strategy for remote manipulation of diverse types of target neurons in the mammalian central nervous system.

neuroscience↗

bioRxiv2023

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 4-6. However, structural screening and phylogenetics are in their infancy, particularly for twilight zone proteins. 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 7-10. However, very few 7TMICs have been identified in non-animal, non-plant taxa. Moreover, these proteins remarkable sequence dissimilarity made it uncertain if 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.

evolutionary biology↗

bioRxiv2023

Evolution of circadian behavioral plasticity through cis-regulatory divergence of a neuropeptide gene

Widely-distributed species experience substantial environmental variation, which they often accommodate through behavioral plasticity. Although this ability is integral to fitness, we have little understanding of the mechanistic basis by which plasticity evolves. One factor that varies seasonally and by latitude is photoperiod (day length). Many organisms, including the cosmopolitan Drosophila melanogaster display circadian plasticity, adjusting to fluctuating photoperiod by varying the timing of their activity to coincide with changing dawn/dusk intervals1. Here, we compare D. melanogaster with the closely-related ecological specialist Drosophila sechellia, an equatorial island endemic that experiences minimal photoperiod variation, to investigate the molecular-genetic basis of circadian plasticity evolution2,3. We discover that D. sechellia displays exceptionally little circadian plasticity compared to D. melanogaster and other non-equatorial drosophilids. Through a screen of circadian mutants in D. melanogaster/D. sechellia hybrids, we identify a role of the neuropeptide Pigment-dispersing factor (Pdf) in this loss. While the coding sequence of Pdf is conserved, we show that Pdf has undergone cis-regulatory divergence in these drosophilids. We document species-specific temporal dynamic properties of Pdf RNA and protein expression, as well as Pdf neuron morphological plasticity, and demonstrate that modulating Pdf expression in D. melanogaster can influence the degree of behavioral plasticity. Furthermore, we find that the Pdf regulatory region exhibits signals of selection across populations of D. melanogaster from different latitudes. Finally, we provide evidence that plasticity confers a selective advantage for D. melanogaster at higher latitudes, while D. sechellia likely suffers fitness costs through reduced copulation success outside its range. Our work defines Pdf as a locus of evolution for circadian plasticity, which might have contributed to both D. melanogasters global distribution and D. sechellias habitat specialization. Moreover, together with spatial changes in Pdf expression reported in high-latitude drosophilid species4,5, our findings highlight this neuropeptide gene as a hotspot for circadian plasticity evolution.

evolutionary biology↗

bioRxiv2023

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.

neuroscience↗

bioRxiv2022

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 ligand-gated ion channels (termed here 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.

evolutionary biology↗

bioRxiv2022

Odor-gated oviposition behavior in an ecological specialist

Colonization of a novel ecological niche can require, or be driven by, evolution of an animals behaviors promoting their reproductive success in the new environment. Little is known about the underlying mechanisms. We have exploited an emerging genetic model for behavioral neuroecology, Drosophila sechellia - a close relative of Drosophila melanogaster that exhibits extreme specialism for Morinda citrifolia noni fruit - to study the evolution and sensory basis of oviposition. D. sechellia produces fewer eggs compared to other drosophilids, but lays these almost exclusively on noni substrates, contrasting with avoidance or indifference of noni by generalist species. Visual, textural and social cues do not explain the species-specificity of this preference. By contrast, loss of olfactory input in D. sechellia, but not D. melanogaster, essentially abolishes egg-laying, suggesting that this sensory modality gates gustatory-driven noni preference. We find the noni bouquet is detected by redundant olfactory pathways. By parsing the fruits volatile chemicals and genetic perturbation of individual olfactory pathways in D. sechellia, we discover a key role for hexanoic acid and its cognate receptor, the Ionotropic receptor 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 oviposition behavior during D. sechellias host specialization.

animal behavior and cognition↗

bioRxiv2022

Comparative transcriptomics of the Drosophila olfactory subsystems identifies a support cell-expressed Osiris protein required for pheromone sensing

The nose of most animals comprises multiple sensory subsystems, which are defined by the expression of different olfactory receptor families. Drosophila melanogaster antennae comprise 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. 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 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 cell-type expression, revealing substantial molecular heterogeneity in the non-neuronal cellular components of these olfactory subsystems. We characterize one Or subsystem-specific non-neuronal molecule, Osiris 8 (Osi8), a conserved member of a large family of insect transmembrane proteins. Osi8 is expressed in tormogen support cells that are associated with pheromone sensing neurons. Loss of Osi8 abolishes high sensitivity neuronal responses to pheromone ligands. Together this work identifies a new protein required for insect pheromone detection, emphasizes the importance of support cells in sensory responses, and provides a resource for future characterization of other olfactory subsystem-specific genes.

neuroscience↗

bioRxiv2021

Odorant receptor copy number change, co-expression, and positive selection establish peripheral coding differences between fly species

1.Despite numerous examples of chemoreceptor gene family expansions and contractions, how these changes relate to modifications in the neural circuitry in which they are expressed remains unclear. Drosophilas Odorant receptor (Or) family is ideal for addressing this question because a large majority of Ors are uniquely expressed in single olfactory sensory neuron (OSN) types. Between-species changes in Or copy number, therefore, may indicate diversification/reduction of OSN populations. To test this, we investigated a rapidly duplicated/deleted subfamily (named Or67a) in Drosophila melanogaster and its sister species (D. simulans, D. sechellia, and D. mauritiana). We found that the common ancestor had three Or67a paralogs that had already diverged adaptively in their odor-evoked responses. Following their speciation, two Or67a paralogs were lost independently in D. melanogaster and D. sechellia, with ongoing positive selection acting on the intact genes. Instead of the expected singular expression of each diverged Ors, the three D. simulans Or67a paralogs are co-expressed. Thus, while neuroanatomy is conserved between these species, independent selection on co-expressed receptors has contributed to species-specific peripheral coding of olfactory information. This work reveals a model of adaptive change previously not considered for olfactory evolution and raises the possibility that similar processes may be operating among the largely uninvestigated cases of Or co-expression.

evolutionary biology↗