The color pattern inducing gene wingless is expressed in specific cell types of campaniform sensilla of a polka-dotted fruit fly, Drosophila guttifera.

A polka-dotted fruit fly, Drosophila guttifera, has a unique pigmentation pattern on its wings and is used as a model for evo-devo studies exploring the mechanism of evolutionary gain of novel traits. In this species, a morphogen-encoding gene, wingless, is expressed in species-specific positions and induces a unique pigmentation pattern. To produce some of the pigmentation spots on wing veins, wingless is thought to be expressed in developing campaniform sensillum cells, but it was unknown which of the four cell types there express(es) wingless. Here we show that two of the cell types, dome cells and socket cells, express wingless, as indicated by in situ hybridization together with immunohistochemistry. This is a unique case in which non-neuronal SOP (sensory organ precursor) progeny cells produce Wingless as an inducer of pigmentation pattern formation. Our finding opens a path to clarifying the mechanism of evolutionary gain of a unique wingless expression pattern by analyzing gene regulation in dome cells and socket cells.

Evidence for the cholinergic markers ChAT and vAChT in sensory cells of the developing antennal nervous system of the desert locust Schistocerca gregaria.

Sensory and motor systems in insects with hemimetabolous development must be ready to mediate adaptive behavior directly on hatching from the egg. For the desert locust S. gregaria, cholinergic transmission from antennal sensillae to olfactory or mechanosensory centers in the brain requires that choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (vAChT) already be present in sensory cells in the first instar. In this study, we used immunolabeling to demonstrate that ChAT and vAChT are both expressed in sensory cells from identifiable sensilla types in the immature antennal nervous system. We observed ChAT expression in dendrites, neurites and somata of putative basiconic-type sensillae at the first instar stage. We also detected vAChT in the sensory axons of these sensillae in a major antennal nerve tract. We then examined whether evidence for cholinergic transmission is present during embryogenesis. Immunolabeling confirms that vAChT is expressed in somata typical of campaniform sensillae, as well as in small sensory cell clusters typically associated with either a large basiconic or coeloconic sensilla, at 99% of embryogenesis. The vAChT is also expressed in the somata of these sensilla types in multiple antennal regions at 90% of embryogenesis, but not at earlier (70%) embryonic stages. Neuromodulators are known to appear late in embryogenesis in neurons of the locust central complex, and the cholinergic system of the antenna may also only reach maturity shortly before hatching.

Sensory neurons that respond to sex and aggregation pheromones in the nymphal cockroach.

In the common pest cockroach, Periplaneta americana, behavioural responses to the sex and aggregation pheromones change in an age-dependent manner. Nymphs are attracted by the aggregation pheromone periplanolide-E (PLD-E) but not by the sex pheromone periplanone-B (PB) in faeces. Adults display prominent behaviours to PB but not to PLD-E. Despite the significant behavioural differences depending on postembryonic developmental stages, peripheral codings of the sex and aggregation pheromones have not been studied in the nymph of any insects as far as we know. In this study, we morphologically and electrophysiologically identified antennal sensilla that respond to PB and PLD-E in nymphal cockroaches. Although nymphs lacked the sex pheromone-responsive single-walled B (sw-B) sensilla identified in adult males, we found PB-responsive sensory neurons (PB-SNs) within newly identified sw-A2 sensilla, which exhibit different shapes but have the same olfactory pores as sw-B sensilla. Interestingly, PLD-E-responsive sensory neurons (PLD-E-SNs) were also identified in the same sensillar type, but PB and PLD-E were independently detected by different SNs. Both PB-SNs and PLD-E-SNs showed high sensitivity to their respective pheromones. The hemimetabolous insect nymph has an ability to detect these pheromones, suggesting that behaviours elicited by pheromones might be established in brain centres depending on postembryonic development.

Mechanoreceptive sensillum fields at the tarsal tip of insect legs.

Groups of mechanoreceptive sensilla form small sensory fields on the ventral rim of the most distal tarsomeres in insects. Within these fields two or three sensilla are located closely together. Anterior and posterior fields are found in all three pairs of legs with only a few exceptions. The composition, exact location, and morphology of the fields were studied in representative species of several insect orders using light and scanning electron microscopy. There was no obvious correlation between field morphology and insect phylogenetic relationships.

In vivo functional characterisation of pheromone binding protein-1 in the silkmoth, Bombyx mori.

Male moths detect sex pheromones emitted by conspecific females with high sensitivity and specificity by the olfactory sensilla on their antennae. Pheromone binding proteins (PBPs) are highly enriched in the sensillum lymph of pheromone sensitive olfactory sensilla and are supposed to contribute to the sensitivity and selectivity of pheromone detection in moths. However, the functional role of PBPs in moth sex pheromone detection in vivo remains obscure. In the silkmoth, Bombyx mori, female moths emit bombykol as a single attractive sex pheromone component along with a small amount of bombykal that negatively modulates the behavioural responses to bombykol. A pair of olfactory receptor neurons, specifically tuned to bombykol or bombykal, co-localise in the trichodeum sensilla, the sensillum lymph of which contains a single PBP, namely, BmPBP1. We analysed the roles of BmPBP1 using BmPBP1-knockout silkmoth lines generated by transcription activator-like effector nuclease-mediated gene targeting. Electroantennogram analysis revealed that the peak response amplitudes of BmPBP1-knockout male antennae to bombykol and bombykal were significantly reduced by a similar percentage when compared with those of the wild-type males. Our results indicate that BmPBP1 plays a crucial role in enhancing the sensitivity, but not the selectivity, of sex pheromone detection in silkmoths.

Two types of sensory proliferation patterns underlie the formation of spatially tuned olfactory receptive fields in the cockroach Periplaneta americana.

In the cockroach Periplaneta americana, to represent pheromone source in the receptive space, axon terminals of sex pheromone-receptive olfactory sensory neurons (pSNs) are topographically organized within the primary center, the macroglomerulus, according to the peripheral locations of sex pheromone-receptive single walled (sw)-B sensilla. In this study, we sought to determine when and where pSNs emerge in the nymphal antenna. We revealed two different pSN proliferation patterns that underlie the formation of topographic organization in the macroglomerulus. In nymphal antennae, which lack sw-B sensilla, pSNs are identified in the shorter sensilla, termed sw-A sensilla. Because new sw-A sensilla emerge on the proximal antenna at every molt, topographic organization in the macroglomerulus must be formed by adding axon terminals of newly emerged pSNs to the lateral region in the macroglomerulus at each molt. At the final molt, a huge number of new sw-B sensilla appeared throughout the whole antenna. Sw-B sensilla in the proximal part of the adult antenna were newly formed during the last instar stage, whereas those located in the distal antenna were transformed from sw-A sensilla. This transformation was accompanied by an increase in the number of pSNs. Axon terminals of newborn pSNs in new sw-B sensilla were recruited to the lateral part of the macroglomerulus, whereas those of newborn pSNs in transformed sw-B sensilla were recruited to the macroglomerulus according to the sensillar location. These mechanisms enable an increase in sensitivity to sex pheromone in adulthood while retaining the topographic map formed during the postembryonic development.

Olfactory Sensory Neurons of the Asian Longhorned Beetle, Anoplophora glabripennis, Specifically Responsive to its two Aggregation-Sex Pheromone Components.

We performed single-sensillum recordings from male and female antennae of the Asian longhorned beetle, Anoplophora glabripennis, that included as stimuli the two components of this species' aggregation-sex pheromone in addition to various general odorants. We compared the aggregation-sex-pheromone-component responses of olfactory sensory neurons (OSNs) to those of OSNs that responded to a variety of plant-related odorants. In the smooth-tipped, tapered, trichoid sensilla on the most distal antennal flagellomeres nos. 10 or 11 of both males and females, we found OSNs with high-amplitude action potentials that were tuned to the aldehyde and alcohol pheromone components and that did not respond to various plant-related volatiles. Because this OSN type responded to both the alcohol and aldehyde components it cannot be considered to be specifically tuned to either component. These large-spiking OSNs were co-compartmentalized in these sensilla with a second, smaller-spiking OSN responding to plant-related volatiles such as geraniol, citronellal, limonene, 1-octanol, nerol and citral. The large-spiking OSNs thus appear to be a type that will be involved in aggregation-sex pheromone pathways targeting a specific glomerulus in the antennal lobe and in generating pheromone-related behavioral responses in A. glabripennis. In other sensilla located in these distal antennal flagellomeres as well as those located more proximally, i.e., mid-length along the antenna on flagellomere nos. 4-7, we found OSNs in blunt-tipped basiconic sensilla that were responsive to other plant-related volatiles, especially the terpenoids, (E,E)-alpha farnesene, (E)-ß-farnesene, ß-caryophyllene, and eugenol. Some of these terpenoids have been implicated in improving attraction to pheromone-baited traps. Some of these same OSNs responded additionally to either of the two sex pheromone components, but because these OSNs also responded to some of the above plant volatiles as shown by cross-adaptation experiments, these OSNs will not be the types that convey sex-pheromone-specific information to the antennal lobe.

Antennal scales improve signal detection efficiency in moths.

The elaborate bipectinate antennae of male moths are thought to increase their sensitivity to female sex pheromones, and so should be favoured by selection. Yet simple filamentous antennae are the most common structure among moths. The stereotypic arrangements of scales on the surface of antennae may resolve this paradox. We use computational fluid dynamics techniques to model how scales on the filamentous antennae of moths affect the passage of different particles in the airflow across the flagellum in both small and large moths. We found that the scales provide an effective solution to improve the efficacy of filamentous antennae, by increasing the concentration of nanoparticles, which resemble pheromones, around the antennae. The smaller moths have a greater increase in antennal efficiency than larger moths. The scales also divert microparticles, which resemble dust, away from the antennal surface, thereby reducing contamination. The positive correlations between antennal scale angles and sensilla number across Heliozelidae moths are consistent with the predictions of our model.

Morphological and electrophysiological differences in tarsal chemosensilla between the wild silkmoth Bombyx mandarina and the domesticated species Bombyx mori.

Gustatory and olfactory senses of phytophagous insects play important roles in the recognition of host plants. In the domestic silkmoth Bombyx mori and its wild species Bombyx mandarina, the morphologies and responses of adult olfactory organs (antennae) have been intensely investigated. However, little is known about these features of adult gustatory organs and the influence of domestication on the gustatory sense. Here we revealed that both species have two types of sensilla (thick [T] and slim [S] types) on the fifth tarsomeres of the adult legs. In both species, females have 3.6-6.9 times more T-sensilla than males. Therefore, T-sensilla seem to play more important roles in females than in males. Moreover, gustatory cells of T-sensilla of B. mandarina females responded intensely to mulberry leaf extract in electrophysiological experiments, while T-sensilla of B. mori females (N4 strain) hardly responded to mulberry leaf extract. These results suggest that T-sensilla of B. mandarina females are involved in the recognition of oviposition sites. We also observed that, in three B. mori strains (N4, p50T, and Kinshu × Showa), the densities of sensilla on the fifth tarsomeres were much lower than in B. mandarina. These results indicate that domestication has influenced the tarsal gustatory system of B. mori.

A map of sensilla and neurons in the taste system of drosophila larvae.

In Drosophila melanogaster larvae, the prime site of external taste reception is the terminal organ (TO). Though investigation on the TO's implications in taste perception has been expanding rapidly, the sensilla of the TO have been essentially unexplored. In this study, we performed a systematic anatomical and molecular analysis of the TO. We precisely define morphological types of TO sensilla taking advantage of volume electron microscopy and 3D image analysis. We corroborate the presence of five external types of sensilla: papilla, pit, spot, knob, and modified papilla. Detailed 3D analysis of their structural organization allowed a finer discrimination into subtypes. We classify three subtypes of papilla and pit sensilla, respectively, and two subtypes of knob sensilla. Further, we determine the repertoire of receptor genes for each sensillum by analyzing GAL4 driver lines of Ir, Gr, Ppk, and Trp receptor genes. We construct a map of the TO, in which the receptor genes are mapped to neurons of individual sensilla. While modified papillum and spot sensilla are not labeled by any GAL4 driver, neurons of the pit, papilla, and knob type are labeled by partially overlapping but different subsets of GAL4 driver lines of the Ir, Gr, and Ppk gene family. The results suggest that pit, papilla and knob sensilla act in contact chemosensation. However, they likely do these employing different stimulus transduction mechanisms to sense the diverse chemicals of their environment.

The sensory arrays of the ant, Temnothorax rugatulus.

Individual differences in response thresholds to task-related stimuli may be one mechanism driving task allocation among social insect workers. These differences may arise at various stages in the nervous system. We investigate variability in the peripheral nervous system as a simple mechanism that can introduce inter-individual differences in sensory information. In this study we describe size-dependent variation of the compound eyes and the antennae in the ant Temnothorax rugatulus. Head width in T. rugatulus varies between 0.4 and 0.7 mm (2.6-3.8 mm body length). But despite this limited range of worker sizes we find sensory array variability. We find that the number of ommatidia and of some, but not all, antennal sensilla types vary with head width. The antennal array of T. rugatulus displays the full complement of sensillum types observed in other species of ants, although at much lower quantities than other, larger, studied species. In addition, we describe what we believe to be a new type of sensillum in hymenoptera that occurs on the antennae and on all body segments. T. rugatulus has apposition compound eyes with 45-76 facets per eye, depending on head width, with average lens diameters of 16.5 µm, rhabdom diameters of 5.7 µm and inter-ommatidial angles of 16.8°. The optical system of T. rugatulus ommatidia is severely under focussed, but the absolute sensitivity of the eyes is unusually high. We discuss the functional significance of these findings and the extent to which the variability of sensory arrays may correlate with task allocation.

A mechanosensory receptor required for food texture detection in Drosophila.

Textural properties provide information on the ingestibility, digestibility and state of ripeness or decay of sources of nutrition. Compared with our understanding of the chemosensory assessment of food, little is known about the mechanisms of texture detection. Here we show that Drosophila melanogaster can discriminate food texture, avoiding substrates that are either too hard or too soft. Manipulations of food substrate properties and flies' chemosensory inputs indicate that texture preferences are revealed only in the presence of an appetitive stimulus, but are not because of changes in nutrient accessibility, suggesting that animals discriminate the substrates' mechanical characteristics. We show that texture preference requires NOMPC, a TRP-family mechanosensory channel. NOMPC localizes to the sensory dendrites of neurons housed within gustatory sensilla, and is essential for their mechanosensory-evoked responses. Our results identify a sensory pathway for texture detection and reveal the behavioural integration of chemical and physical qualities of food.

Scanning Electron Microscopy Study of the Antennal Sensilla of Monema flavescens Walker (Lepidoptera: Limacodidae).

Monema flavescens Walker (Lepidoptera: Limacodidae) is a serious polyphagous defoliator. Using scanning electron microscopy, the external morphology of the antennal sensilla of this pest was examined for a better understanding of the mechanisms of insect-insect and insect-plant chemical communications. The antennae of M. flavescens were filiform in shape, and 11 morphological types of sensilla were found in both sexes. Six types of likely chemosensory sensilla were identified: uniporous sensilla chaetica, multiporous sensilla trichodea, and four types of multiporous sensilla basiconica. The sensilla identified as likely mechanoreceptors included two subtypes of aporous sensilla chaetica, aporous sensilla coeloconica, aporous sensilla styloconica, and Böhm's bristles, whereas the position of the antennae was monitored by Böhm's bristles.

Sensory Trichites Associated With the Food Canal of Chrysops callidus (Diptera: Tabanidae).

Twelve pairs of putative sensory trichites (= sensilla) in the food canal of Chrysops callidus Osten Sacken are described. Eight paired trichites are located in the distal half of the food canal of all flies in the sample population (n = 26 females), but paired trichites 9 through 12 were found in variable numbers in the proximal half of the canal. Trichites were more closely spaced in the distal half of the food canal, and more widely spaced in the proximal half of the canal, indicating that monitoring blood flow is more critical in the distal region of the canal. Moreover, trichites were not precisely paired, with mean position for each right wall trichite 1 through 8 being slightly anterior to its counterpart in the left wall. A short, funnel-shaped vestibule was evident at the labrum terminus, opening distally to the outside and proximally into the food canal (the vestibule/food canal junction). There were two pairs of sensilla (one pair of basicone design, the other setiform) observed at the base of the vestibule just anterior to the junction of the vestibule with the food canal. These vestibular sensilla were constant in type, number and position, in every member of the sample population.

A re-examination of the selection of the sensory organ precursor of the bristle sensilla of Drosophila melanogaster.

The bristle sensillum of the imago of Drosophila is made of four cells that arise from a sensory organ precursor cell (SOP). This SOP is selected within proneural clusters (PNC) through a mechanism that involves Notch signalling. PNCs are defined through the expression domains of the proneural genes, whose activities enables cells to become SOPs. They encode tissue specific bHLH proteins that form functional heterodimers with the bHLH protein Daughterless (Da). In the prevailing lateral inhibition model for SOP selection, a transcriptional feedback loop that involves the Notch pathway amplifies small differences of proneural activity between cells of the PNC. As a result only one or two cells accumulate sufficient proneural activity to adopt the SOP fate. Most of the experiments that sustained the prevailing lateral inhibition model were performed a decade ago. We here re-examined the selection process using recently available reagents. Our data suggest a different picture of SOP selection. They indicate that a band-like region of proneural activity exists. In this proneural band the activity of the Notch pathway is required in combination with Emc to define the PNCs. We found a sub-group in the PNCs from which a pre-selected SOP arises. Our data indicate that most imaginal disc cells are able to adopt a proneural state from which they can progress to become SOPs. They further show that bristle formation can occur in the absence of the proneural genes if the function of emc is abolished. These results suggest that the tissue specific proneural proteins of Drosophila have a similar function as in the vertebrates, which is to determine the time of emergence and position of the SOP and to stabilise the proneural state.

Mechanosensory interactions drive collective behaviour in Drosophila.

Collective behaviour enhances environmental sensing and decision-making in groups of animals. Experimental and theoretical investigations of schooling fish, flocking birds and human crowds have demonstrated that simple interactions between individuals can explain emergent group dynamics. These findings indicate the existence of neural circuits that support distributed behaviours, but the molecular and cellular identities of relevant sensory pathways are unknown. Here we show that Drosophila melanogaster exhibits collective responses to an aversive odour: individual flies weakly avoid the stimulus, but groups show enhanced escape reactions. Using high-resolution behavioural tracking, computational simulations, genetic perturbations, neural silencing and optogenetic activation we demonstrate that this collective odour avoidance arises from cascades of appendage touch interactions between pairs of flies. Inter-fly touch sensing and collective behaviour require the activity of distal leg mechanosensory sensilla neurons and the mechanosensory channel NOMPC. Remarkably, through these inter-fly encounters, wild-type flies can elicit avoidance behaviour in mutant animals that cannot sense the odour--a basic form of communication. Our data highlight the unexpected importance of social context in the sensory responses of a solitary species and open the door to a neural-circuit-level understanding of collective behaviour in animal groups.

Physiological organization and topographic mapping of the antennal olfactory sensory neurons in female hawkmoths, Manduca sexta.

The hawkmoth, Manduca sexta, has been a keystone system for developmental, neurobiological, and ecological studies for several decades. Because many of its behaviors are driven by olfactory cues, a thorough understanding of the Manduca olfactory system is essential to studying its biology. With the aim of functionally characterizing single antennal olfactory sensory neurons (OSNs) and determining their detailed topographic location, we performed systematic single-sensillum recordings on 4 morphological types of olfactory sensilla: trichoid-A and -B and basiconic-A and -B. We were able to unambiguously differentiate the colocalized cells associated with single sensilla based on their spike amplitudes. Using a panel of 61 biologically relevant compounds established in behavioral and gas chromatography-electrophysiology experiments, we made 223 recordings from these sensilla. Based on the response spectra of 187 responding OSNs, the sensilla fell into 12 distinct functional classes encompassing 29 OSNs. Selectivity of the 25 responding OSNs varied from narrowly tuned (responding to only one or a subset of compounds), to very broadly tuned (responding to multiple compounds), in a concentration-dependent manner. Four OSNs, however, did not respond to the tested components. Topographic mapping of the sensilla revealed that some physiological sensillum types are confined to particular locations on the antennal surface while other classes are more or less irregularly scattered all over the antennal annuli. Such information will prove beneficial for future receptor deorphanization, in situ hybridization, and molecular manipulation experiments.

Antennal olfactory sensilla responses to insect chemical repellents in the common bed bug, Cimex lectularius.

Populations of the common bed bug Cimex lectularius (Hemiptera; Cimicidae), a temporary ectoparasite on both humans and animals, have surged in many developed countries. Similar to other haematophagous arthropods, C. lectularius relies on its olfactory system to detect semiochemicals in the environment, including both attractants and repellents. To elucidate the olfactory responses of the common bed bug to commonly used insect chemical repellents, particularly haematophagous repellents, we investigated the neuronal responses of individual olfactory sensilla in C. lectularius' antennae to 52 insect chemical repellents, both synthetic and botanic. Different types of sensilla displayed highly distinctive response profiles. While C sensilla did not respond to any of the insect chemical repellents, Dγ sensilla proved to be the most sensitive in response to terpene-derived insect chemical repellents. Different chemical repellents elicited neuronal responses with differing temporal characteristics, and the responses of the olfactory sensilla to the insect chemical repellents were dose-dependent, with an olfactory response to the terpene-derived chemical repellent, but not to the non-terpene-derived chemical repellents. Overall, this study furnishes a comprehensive map of the olfactory response of bed bugs to commonly used insect chemical repellents, providing useful information for those developing new agents (attractants or repellents) for bed bug control.

Chambered cuticle, pellicles, strange sensilla, and extraordinary muscle arrangements: a study of the micropterigid larval trunk (Lepidoptera: Micropterigidae).

The larvsal trunk wall of Sabatinca chalcophanes (Meyrick, 1885), representing the "sabatincoid morphotype," is described (brightfield and polarization microscopies, scanning and transmission electron microscopies). Eight sensillum types are identified, including four previously undescribed subventral and ventral kinds. The cuticle is nonsolid, the exocuticle being chambered in a honeycomb-like fashion with chamber walls apparently secreted along epidermal cells boundaries. The chamber contents open to the exterior via minute pores in the chamber roofs. A space between endo and exocuticle communicates with the chamber interiors via pores in the chamber floors; the dense endocuticular surface in places form thickened domes. On the lower trunk region, lateral chamber walls are highly porous (lattice like), hence their contents are continuous; individual chamber roofs here are markedly convex, and the external trunk surface, therefore, papillate. The trunk surface is more or less completely covered by a pellicle, likely formed by exudates from the exocuticular chambers. Unusually for Lepidoptera all trunk muscles are slender strands covering a modest proportion of the inner trunk surface. Conspicuous insertion "nodes" are located at lateral and ventral segmental boundaries, ventromedially near segmental midlengths, and paramedially on the dorsum behind segmental midlengths. Overall similar cuticular specializations are also present in the distantly related Micropterix, strongly supporting micropterigid monophyly.

The antennal lobe of Libellula depressa (Odonata, Libellulidae).

Here we describe the antennal lobe of Libellula depressa (Odonata, Libellulidae), identified on the basis of the projections of the afferent sensory neurons stemming from the antennal flagellum sensilla. Immunohistochemical neuropil staining as well as antennal backfills revealed sensory neuron terminal arborizations covering a large portion of the antennal lobe. No clear glomerular structure was identified, thus suggesting an aglomerular antennal lobe condition as previously reported in Palaeoptera. The terminal arbors of backfilled sensory neurons do, however, form spherical knots, probably representing the connections between the few afferent neurons and the antennal lobe interneurons. The reconstruction revealed that the proximal part of the antennal nerve is divided into two branches that innervate two spatially separated areas of the antennal lobe, an anterioventral lobe and a larger posteriodorsal lobe. Our data are consistent with the hypothesis that one tract of the antennal nerve of L. depressa contains olfactory sensory neurons projecting into one of the sublobes, while the other tract contains thermo-hygroreceptive neurons projecting into the other sublobe.