The Touching Tail of a Mechanotransduction Channel.

In mechanotransduction, sensory receptors convert force into electrical signals to mediate such diverse functions as touch, pain, and hearing. In this issue of Cell, Zhang et al. present evidence that the fly NompC channel senses mechanical stimuli using its N-terminal tail as a tether between the cell membrane and microtubules.

A genome-wide Drosophila screen for heat nociception identifies α2δ3 as an evolutionarily conserved pain gene.

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.

Hunting with heat: thermosensory-driven foraging in mosquitoes, snakes and beetles.

Animals commonly use thermosensation, the detection of temperature and its variation, for defensive purposes: to maintain appropriate body temperature and to avoid tissue damage. However, some animals also use thermosensation to go on the offensive: to hunt for food. The emergence of heat-dependent foraging behavior has been accompanied by the evolution of diverse thermosensory organs of often exquisite thermosensitivity. These organs detect the heat energy emitted from food sources that range from nearby humans to trees burning in a forest kilometers away. Here, we examine the biophysical considerations, anatomical specializations and molecular mechanisms that underlie heat-driven foraging. We focus on three groups of animals that each meet the challenge of detecting heat from potential food sources in different ways: (1) disease-spreading vector mosquitoes, which seek blood meals from warm-bodied hosts at close range, using warming-inhibited thermosensory neurons responsive to conductive and convective heat flow; (2) snakes (vipers, pythons and boas), which seek warm-blooded prey from ten or more centimeters away, using warmth-activated thermosensory neurons housed in an organ specialized to harvest infrared radiation; and (3) fire beetles, which maximize their offspring's feeding opportunities by seeking forest fires from kilometers away, using mechanosensory neurons housed in an organ specialized to convert infrared radiation into mechanosensory stimuli. These examples highlight the diverse ways in which animals exploit the heat emanating from potential food sources, whether this heat reflects ongoing metabolic activity or a recent lightning strike, to secure a nutritious meal for themselves or for their offspring.

A gustatory receptor paralogue controls rapid warmth avoidance in Drosophila.

Behavioural responses to temperature are critical for survival, and animals from insects to humans show strong preferences for specific temperatures. Preferred temperature selection promotes avoidance of adverse thermal environments in the short term and maintenance of optimal body temperatures over the long term, but its molecular and cellular basis is largely unknown. Recent studies have generated conflicting views of thermal preference in Drosophila, attributing importance to either internal or peripheral warmth sensors. Here we reconcile these views by showing that thermal preference is not a singular response, but involves multiple systems relevant in different contexts. We found previously that the transient receptor potential channel TRPA1 acts internally to control the slowly developing preference response of flies exposed to a shallow thermal gradient. We now find that the rapid response of flies exposed to a steep warmth gradient does not require TRPA1; rather, the gustatory receptor GR28B(D) drives this behaviour through peripheral thermosensors. Gustatory receptors are a large gene family, widely studied in insect gustation and olfaction, and are implicated in host-seeking by insect disease vectors, but have not previously been implicated in thermosensation. At the molecular level, GR28B(D) misexpression confers thermosensitivity upon diverse cell types, suggesting that it is a warmth sensor. These data reveal a new type of thermosensory molecule and uncover a functional distinction between peripheral and internal warmth sensors in this tiny ectotherm reminiscent of thermoregulatory systems in larger, endothermic animals. The use of multiple, distinct molecules to respond to a given temperature, as observed here, may facilitate independent tuning of an animal's distinct thermosensory responses.

Sensory determinants of behavioral dynamics in Drosophila thermotaxis.

Complex animal behaviors are built from dynamical relationships between sensory inputs, neuronal activity, and motor outputs in patterns with strategic value. Connecting these patterns illuminates how nervous systems compute behavior. Here, we study Drosophila larva navigation up temperature gradients toward preferred temperatures (positive thermotaxis). By tracking the movements of animals responding to fixed spatial temperature gradients or random temperature fluctuations, we calculate the sensitivity and dynamics of the conversion of thermosensory inputs into motor responses. We discover three thermosensory neurons in each dorsal organ ganglion (DOG) that are required for positive thermotaxis. Random optogenetic stimulation of the DOG thermosensory neurons evokes behavioral patterns that mimic the response to temperature variations. In vivo calcium and voltage imaging reveals that the DOG thermosensory neurons exhibit activity patterns with sensitivity and dynamics matched to the behavioral response. Temporal processing of temperature variations carried out by the DOG thermosensory neurons emerges in distinct motor responses during thermotaxis.

Running hot and cold: behavioral strategies, neural circuits, and the molecular machinery for thermotaxis in C. elegans and Drosophila.

Like other ectotherms, the roundworm Caenorhabditis elegans and the fruit fly Drosophila melanogaster rely on behavioral strategies to stabilize their body temperature. Both animals use specialized sensory neurons to detect small changes in temperature, and the activity of these thermosensors governs the neural circuits that control migration and accumulation at preferred temperatures. Despite these similarities, the underlying molecular, neuronal, and computational mechanisms responsible for thermotaxis are distinct in these organisms. Here, we discuss the role of thermosensation in the development and survival of C. elegans and Drosophila, and review the behavioral strategies, neuronal circuits, and molecular networks responsible for thermotaxis behavior.

Modulation of TRPA1 thermal sensitivity enables sensory discrimination in Drosophila.

Discriminating among sensory stimuli is critical for animal survival. This discrimination is particularly essential when evaluating whether a stimulus is noxious or innocuous. From insects to humans, transient receptor potential (TRP) channels are key transducers of thermal, chemical and other sensory cues. Many TRPs are multimodal receptors that respond to diverse stimuli, but how animals distinguish sensory inputs activating the same TRP is largely unknown. Here we determine how stimuli activating Drosophila TRPA1 are discriminated. Although Drosophila TRPA1 responds to both noxious chemicals and innocuous warming, we find that TRPA1-expressing chemosensory neurons respond to chemicals but not warmth, a specificity conferred by a chemosensory-specific TRPA1 isoform with reduced thermosensitivity compared to the previously described isoform. At the molecular level, this reduction results from a unique region that robustly reduces the channel's thermosensitivity. Cell-type segregation of TRPA1 activity is critical: when the thermosensory isoform is expressed in chemosensors, flies respond to innocuous warming with regurgitation, a nocifensive response. TRPA1 isoform diversity is conserved in malaria mosquitoes, indicating that similar mechanisms may allow discrimination of host-derived warmth--an attractant--from chemical repellents. These findings indicate that reducing thermosensitivity can be critical for TRP channel functional diversification, facilitating their use in contexts in which thermal sensitivity can be maladaptive.

Analysis of Drosophila TRPA1 reveals an ancient origin for human chemical nociception.

Chemical nociception, the detection of tissue-damaging chemicals, is important for animal survival and causes human pain and inflammation, but its evolutionary origins are largely unknown. Reactive electrophiles are a class of noxious compounds humans find pungent and irritating, such as allyl isothiocyanate (in wasabi) and acrolein (in cigarette smoke). Diverse animals, from insects to humans, find reactive electrophiles aversive, but whether this reflects conservation of an ancient sensory modality has been unclear. Here we identify the molecular basis of reactive electrophile detection in flies. We demonstrate that Drosophila TRPA1 (Transient receptor potential A1), the Drosophila melanogaster orthologue of the human irritant sensor, acts in gustatory chemosensors to inhibit reactive electrophile ingestion. We show that fly and mosquito TRPA1 orthologues are molecular sensors of electrophiles, using a mechanism conserved with vertebrate TRPA1s. Phylogenetic analyses indicate that invertebrate and vertebrate TRPA1s share a common ancestor that possessed critical characteristics required for electrophile detection. These findings support emergence of TRPA1-based electrophile detection in a common bilaterian ancestor, with widespread conservation throughout vertebrate and invertebrate evolution. Such conservation contrasts with the evolutionary divergence of canonical olfactory and gustatory receptors and may relate to electrophile toxicity. We propose that human pain perception relies on an ancient chemical sensor conserved across approximately 500 million years of animal evolution.

An internal thermal sensor controlling temperature preference in Drosophila.

Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. Here we identify a small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly's preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly's preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival.

Structural basis of ligand specificity and channel activation in an insect gustatory receptor.

Gustatory receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors, making their structural investigation a vital step toward such applications. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect odorant receptors (ORs). Upon fructose binding, BmGr9's channel gate opens through helix S7b movements. In contrast to ORs, BmGr9's ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also, unlike ORs, fructose binding by BmGr9 involves helix S5 and a pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with different ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

DMKPs provide a generalizable strategy for studying genes required for reproduction or viability in nontraditional model organisms.

The advent of CRISPR/Cas9-mediated genome editing has expanded the range of animals amenable to targeted genetic analysis. This has accelerated research in animals not traditionally studied using molecular genetics. However, studying genes essential for reproduction or survival in such animals remains challenging, as they lack the tools that aid genetic analysis in traditional genetic model organisms. We recently introduced the use of distinguishably marked knock-in pairs (DMKPs) as a strategy for rapid and reliable genotyping in such species. Here we show that DMKPs also facilitate the maintenance and study of mutations that cannot be maintained in a homozygous state, a group which includes recessive lethal and sterile mutations. Using DMKPs, we disrupt the zero population growth locus in Drosophila melanogaster and in the dengue vector mosquito Aedes aegypti. In both species, DMKPs enable the maintenance of zero population growth mutant strains and the reliable recovery of zero population growth mutant animals. Male and female gonad development is disrupted in fly and mosquito zero population growth mutants, rendering both sexes sterile. In Ae. aegypti, zero population growth mutant males remain capable of inducing a mating refractory period in wild-type females and of competing with wild-type males for mates, properties compatible with zero population growth serving as a target in mosquito population suppression strategies. DMKP is readily generalizable to other species amenable to CRISPR/Cas9-mediated gene targeting, and should facilitate the study of sterile and lethal mutations in multiple organisms not traditionally studied using molecular genetics.

Humidity sensors that alert mosquitoes to nearby hosts and egg-laying sites.

To reproduce and to transmit disease, female mosquitoes must obtain blood meals and locate appropriate sites for egg laying (oviposition). While distinct sensory cues drive each behavior, humidity contributes to both. Here, we identify the mosquito's humidity sensors (hygrosensors). Using generalizable approaches designed to simplify genetic analysis in non-traditional model organisms, we demonstrate that the ionotropic receptor Ir93a mediates mosquito hygrosensation as well as thermosensation. We further show that Ir93a-dependent sensors drive human host proximity detection and blood-feeding behavior, consistent with the overlapping short-range heat and humidity gradients these targets generate. After blood feeding, gravid females require Ir93a to seek high humidity associated with preferred egg-laying sites. Reliance on Ir93a-dependent sensors to promote blood feeding and locate potential oviposition sites is shared between the malaria vector Anopheles gambiae and arbovirus vector Aedes aegypti. These Ir93a-dependent systems represent potential targets for efforts to control these human disease vectors.

Structure of an insect gustatory receptor.

Gustatory Receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors. However, GR structures have not been experimentally determined. We present structures of Bombyx mori Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect Olfactory Receptors (ORs). Upon fructose binding, BmGr9's ion channel gate opens through helix S7b movements. In contrast to ORs, BmGR9's ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also unlike ORs, fructose binding by BmGr9 involves helix S5 and a binding pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with distinct ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.

Salt sensing: A new receptor for an ancient taste.

Regulation of salt intake is important for animals from flies to humans. A new study clarifies the molecular receptors mediating attraction to low salt concentrations in Drosophila, suggesting a surprisingly fly-specific solution to the challenge of detecting this universal tastant.

Navigational decision making in Drosophila thermotaxis.

A mechanistic understanding of animal navigation requires quantitative assessment of the sensorimotor strategies used during navigation and quantitative assessment of how these strategies are regulated by cellular sensors. Here, we examine thermotactic behavior of the Drosophila melanogaster larva using a tracking microscope to study individual larval movements on defined temperature gradients. We discover that larval thermotaxis involves a larger repertoire of strategies than navigation in smaller organisms such as motile bacteria and Caenorhabditis elegans. Beyond regulating run length (i.e., biasing a random walk), the Drosophila melanogaster larva also regulates the size and direction of turns to achieve and maintain favorable orientations. Thus, the sharp turns in a larva's trajectory represent decision points for selecting new directions of forward movement. The larva uses the same strategies to move up temperature gradients during positive thermotaxis and to move down temperature gradients during negative thermotaxis. Disrupting positive thermotaxis by inactivating cold-sensitive neurons in the larva's terminal organ weakens all regulation of turning decisions, suggesting that information from one set of temperature sensors is used to regulate all aspects of turning decisions. The Drosophila melanogaster larva performs thermotaxis by biasing stochastic turning decisions on the basis of temporal variations in thermosensory input, thereby augmenting the likelihood of heading toward favorable temperatures at all times.

Distinct TRP channels are required for warm and cool avoidance in Drosophila melanogaster.

The ability to sense and respond to subtle variations in environmental temperature is critical for animal survival. Animals avoid temperatures that are too cold or too warm and seek out temperatures favorable for their survival. At the molecular level, members of the transient receptor potential (TRP) family of cation channels contribute to thermosensory behaviors in animals from flies to humans. In Drosophila melanogaster larvae, avoidance of excessively warm temperatures is known to require the TRP protein dTRPA1. Whether larval avoidance of excessively cool temperatures also requires TRP channel function, and whether warm and cool avoidance use the same or distinct TRP channels has been unknown. Here we identify two TRP channels required for cool avoidance, TRPL and TRP. Although TRPL and TRP have previously characterized roles in phototransduction, their function in cool avoidance appears to be distinct, as neither photoreceptor neurons nor the phototransduction regulators NORPA and INAF are required for cool avoidance. TRPL and TRP are required for cool avoidance; however they are dispensable for warm avoidance. Furthermore, cold-activated neurons in the larvae are required for cool but not warm avoidance. Conversely, dTRPA1 is essential for warm avoidance, but not cool avoidance. Taken together, these data demonstrate that warm and cool avoidance in the Drosophila larva involves distinct TRP channels and circuits.

A divalent boost from magnesium.

Enhanced levels of dietary magnesium improve long-term memory in fruit flies.

Mosquito heat seeking is driven by an ancestral cooling receptor.

Mosquitoes transmit pathogens that kill >700,000 people annually. These insects use body heat to locate and feed on warm-blooded hosts, but the molecular basis of such behavior is unknown. Here, we identify ionotropic receptor IR21a, a receptor conserved throughout insects, as a key mediator of heat seeking in the malaria vector Anopheles gambiae Although Ir21a mediates heat avoidance in Drosophila, we find it drives heat seeking and heat-stimulated blood feeding in Anopheles At a cellular level, Ir21a is essential for the detection of cooling, suggesting that during evolution mosquito heat seeking relied on cooling-mediated repulsion. Our data indicate that the evolution of blood feeding in Anopheles involves repurposing an ancestral thermoreceptor from non-blood-feeding Diptera.

Connectomics Analysis Reveals First-, Second-, and Third-Order Thermosensory and Hygrosensory Neurons in the Adult Drosophila Brain.

Animals exhibit innate and learned preferences for temperature and humidity-conditions critical for their survival and reproduction. Leveraging a whole-brain electron microscopy volume, we studied the adult Drosophila melanogaster circuitry associated with antennal thermo- and hygrosensory neurons. We have identified two new target glomeruli in the antennal lobe, in addition to the five known ones, and the ventroposterior projection neurons (VP PNs) that relay thermo- and hygrosensory information to higher brain centers, including the mushroom body and lateral horn, seats of learned and innate behavior. We present the first connectome of a thermo- and hygrosensory neuropil, the lateral accessory calyx (lACA), by reconstructing neurons downstream of heating- and cooling-responsive VP PNs. A few mushroom body-intrinsic neurons solely receive thermosensory input from the lACA, while most receive additional olfactory and thermo- and/or hygrosensory PN inputs. Furthermore, several classes of lACA-associated neurons form a local network with outputs to other brain neuropils, suggesting that the lACA serves as a hub for thermo- and hygrosensory circuitry. For example, DN1a neurons link thermosensory PNs in the lACA to the circadian clock via the accessory medulla. Finally, we survey strongly connected downstream partners of VP PNs across the protocerebrum; these include a descending neuron targeted by dry-responsive VP PNs, meaning that just two synapses might separate hygrosensory inputs from motor circuits. These data provide a comprehensive first- and second-order layer analysis of Drosophila thermo- and hygrosensory systems and an initial survey of third-order neurons that could directly modulate behavior.