Using Azadirachtin to Transform Spodoptera frugiperda from Pest to Natural Enemy.

Spodoptera frugiperda and Rhopalosiphum maidis, as main pests, seriously harm the safety of maize. At present, chemical pesticides are mainly used to control these pests. However, due to residue and resistance problems, more green, environmentally benign, simple preventive control technology is needed. In this study, we reported the reason for the antifeedant activity of azadirachtin on S. frugiperda and proposed that S. frugiperda treated with azadirachtin would turn from pest into natural enemy. S. frugiperda showed an obvious antifeeding phenomenon to maize leaf treated with various azadirachtin concentrations (0.5~20 mg/L). It was found that maize leaf treated with 1 mg/L of azadirachtin has a stimulating effect on the antenna and sensillum basiconicum of S. frugiperda, and azadirachtin can affect the feeding behavior of S. frugiperda. Additionally, after treating maize leaves or maize leaves + R. maidis with 1 mg/L of azadirachtin, the predatory behavior of S. frugiperda changed from a preference for eating maize leaves to R. maidis. Moreover, the molting of R. maidis can promote the change of this predatory behavior. Our results, for the first time, propose that the combined control technology of azadirachtin insecticide and biological control could turn S. frugiperda from pest into natural enemy, which can effectively eliminate R. maidis and protect maize. This combined control technology provides a new way for pest management and has good ecological, environmental, and economic benefits.

Histamine gustatory aversion in Drosophila melanogaster.

Many foods and drinks contain histamine; however, the mechanisms that drive histamine taste perception have not yet been investigated. Here, we use a simple model organism, Drosophila melanogaster, to dissect the molecular sensors required to taste histamine. We first investigated histidine and histamine taste perception by performing a binary food choice assay and electrophysiology to identify essential sensilla for histamine sensing in the labellum. Histamine was found to activate S-type sensilla, which harbor bitter-sensing gustatory receptor neurons. Moreover, unbiased genetic screening for chemoreceptors revealed that a gustatory receptor, GR22e and an ionotropic receptor, IR76b are required for histamine sensing. Ectopic expression of GR22e was sufficient to induce a response in I-type sensilla, which normally do not respond to histamine. Taken together, our findings provide new insights into the mechanisms by which insects discriminate between the toxic histamine and beneficial histidine via their taste receptors.

Aldehyde-specific responses of olfactory sensory neurons in the praying mantis.

Although praying mantises rely mainly on vision for predatory behaviours, olfaction also plays a critical role in feeding and mating behaviours. However, the receptive processes underlying olfactory signals remain unclear. Here, we identified olfactory sensory neurons (OSNs) that are highly tuned to detect aldehydes in the mantis Tenodera aridifolia. In extracellular recordings from OSNs in basiconic sensilla on the antennae, we observed three different spike shapes, indicating that at least three OSNs are housed in a single basiconic sensillum. Unexpectedly, one of the three OSNs exhibited strong excitatory responses to a set of aldehydes. Based on the similarities of the response spectra to 15 different aldehydes, the aldehyde-specific OSNs were classified into three classes: B, S, and M. Class B broadly responded to most aldehydes used as stimulants; class S responded to short-chain aldehydes (C3-C7); and class M responded to middle-length chain aldehydes (C6-C9). Thus, aldehyde molecules can be finely discriminated based on the activity patterns of a population of OSNs. Because many insects emit aldehydes for pheromonal communication, mantises might use aldehydes as olfactory cues for locating prey habitat.

Antifeeding effects of azadirachtin on the fifth instar Spodoptera litura larvae and the analysis of azadirachtin on target sensilla around mouthparts.

To clarify the types, number, and distribution of sensilla on the head of the fifth instar Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) larvae and identify the main sensilla of azadirachtin acting on larvae, scanning electron microscopy was used to study the morphology of the head and sensilla on the mouthparts. The four sensilla-sensillum basiconicum, sensillum chaeticum, sensillum styloconicum, and sensillum trichodeum-on the head of the fifth instar larvae were treated with 0, 0.1, 0.5, 1, 2, and 4 mg/kg azadirachtin by a microdrop method. The larvae showed an obvious antifeeding effect with azadirachtin. And higher the concentration of azadirachtin, the more obvious the phenomenon of antifeeding activity. The sensillum styloconicum and the sensillum trichodeum were the main sensilla for azadirachtin. When 1 mg/kg azadirachtin was used to treat sensillum styloconicum and sensillum basiconicum, the fifth instar larvae of S. litura showed obvious antifeedant activity and the cumulative feed intake for 24 hr was no more than 30% of the leaf area. Quantitative reverse-transcription polymerase chain reaction verified the expression patterns of some Grs, indicating that Grst43a was upregulated by 1.3- and 3.9-fold, Gor24 was upregulated by 2.5- and 3.3-fold, Gr5a was downregulated by 0.6-fold and upregulated by 2.0-fold, and Gr28a was downregulated by 0.8-fold and upregulated by 3.6-fold upon treatment with 0.5 mg/kg and 1 mg/kg azadirachtin in 24 hr. Gr genes participated in the identification of bitterness and we speculated that Gr genes may indirectly lead to the occurrence of antifeeding behavior.

Characterization of olfactory sensory neurons in the red clover seed weevil, Protapion trifolii (Coleoptera: Brentidae) and comparison to the closely related species P. fulvipes.

Protapion trifolii and P. fulvipes (Coleoptera: Brentidae) are major pests in European clover seed production. Previous studies have reported a high host plant fidelity of these weevils for red and white clover species, respectively, driven by host plant olfactory cues. Given the specific host preferences observed in these weevils, we aimed to elucidate to which extent such selectivity is reflected in their peripheral olfactory systems. Using an electrophysiological approach, we performed the first functional characterisation of olfactory sensory neurons (OSNs) in P. trifolii to a panel of volatile compounds emitted by red clover plants, and compared the results with the reported OSN types of P. fulvipes. Nineteen OSN classes were characterized in P. trifolii, with the majority of these neurons responding strongly to common volatiles released by the host plant. Based on response profiles, eight of these OSN classes have clear matches to OSN classes in P. fulvipes. The OSN colocalisation patterns and antennal frequency of these classes are similar in the two species. Additionally, the responses of these OSNs are generally highly conserved in the two species, with clear response shifts only revealed for two of the OSN classes. These response shifts in combination with additional response dissimilarities for compounds that vary in abundance between red and white clover plants may underlie the species-specific host preferences. Further behavioural and field experiments should focus on these differentially detected compounds to elucidate their potential role in host selection and use in semiochemical-based control of these pests.

Comparison of male antennal morphology and sensilla physiology for sex pheromone olfactory sensing between sibling moth species: Ectropis grisescens and Ectropis obliqua (Geometridae).

Ectropis grisescens and Ectropis obliqua (Lepidoptera: Geometridae) are sibling pest species that co-occur on tea plants. The sex pheromone components of both species contain (Z,Z,Z)-3,6,9-octadecatriene and (Z,Z)-3,9-cis-6,7-epoxy-octadecadiene. E. obliqua has (Z,Z)-3,9-cis-6,7-epoxy-nonadecadiene as an additional sex pheromone component, which ensures reproductive segregation between the two species. To ascertain the detection mechanism of olfactory organs for sex pheromone components of E. grisescens and E. obliqua, we applied scanning electron microscopy and single sensillum recording to compare antennal morphology and sensillum physiology in the two species. There was no apparent morphological difference between the antennae of the two species. Both species responded similarly to all three sex pheromone components, including, E. obliqua specific component. The distribution patterns of antennal sensilla trichodea differed between the two species. Sex pheromone olfactory sensing in these sibling species appears to be determined by the density of different types of olfactory sensing neurons. Dose-dependent responses of sensilla trichodea type 1 to (Z,Z)-3,9-cis-6,7-epoxy-octadecadiene, the most abundant component, showed an "all or none" pattern and the other two components showed sigmoidal dose-response curves with a half threshold of 10-4 (dilution equal to the concentration of 10 µg/µl). These results suggest that the major sex pheromone component functions as an on-off controller while secondary components function as modulators during olfactory transmission to the primary olfactory center.

Neuronal Responses of Antennal Olfactory Sensilla to Insect Chemical Repellents in the Yellow Fever Mosquito, Aedes aegypti.

The yellow fever mosquito, Aedes aegypti, is a vector of many human diseases such as yellow fever, dengue fever, and Zika. As insecticide resistance has been widely reported, chemical repellents have been adopted as alternative options for mosquito and mosquito-borne disease control. This study characterized the responses of olfactory receptor neurons (ORNs) in different types of antennal olfactory sensilla in Ae. aegypti to 48 chemicals that exhibited repellent activity in various insect species. Both excitatory and inhibitory responses were observed from ORNs in response to these chemicals and differential tuning properties were also observed among ORNs. Remarkable excitatory responses were recorded from the ORNs in sensilla SST1, SST2, SBTI, SBTII, and LST2, while inhibitory activities were detected from a neuron in sensillum SST2 in response to several terpene/terpenoid compounds. Moreover, the temporal dynamics of neuronal responses were found to be compound-specific and concentration-dependent. Hierarchical cluster analysis and principal component analysis of the response to each compound across ORNs in seven types of olfactory sensilla in Ae. aegypti revealed that odor reception depended not only on chemical class but also specific chemical structure. Results of this study give new insights into the sensory physiology of Aedes mosquitoes to the chemical repellents and should contribute to the development of new repellent reagents for human protection.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility.

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Chemosensory Responses to the Repellent Nepeta Essential Oil and Its Major Component Nepetalactone by Aedes aegypti (Diptera: Culicidae), a Vector of Zika Virus.

Nepeta essential oil (Neo; catnip) and its major component, nepetalactone, have long been known to repel insects including mosquitoes. However, the neural mechanisms through which these repellents are detected by mosquitoes, including the yellow fever mosquito Aedes aegypti (L.), an important vector of Zika virus, were poorly understood. Here we show that Neo volatiles activate olfactory receptor neurons within the basiconic sensilla on the maxillary palps of female Ae. aegypti. A gustatory receptor neuron sensitive to the feeding deterrent quinine and housed within sensilla on the labella of females was activated by both Neo and nepetalactone. Activity of a second gustatory receptor neuron sensitive to the feeding stimulant sucrose was suppressed by both repellents. Our results provide neural pathways for the reported spatial repellency and feeding deterrence of these repellents. A better understanding of the neural input through which female mosquitoes make decisions to feed will facilitate design of new repellents and management strategies involving their use.

Mode of Action of the Natural Insecticide, Decaleside Involves Sodium Pump Inhibition.

Decalesides are a new class of natural insecticides which are toxic to insects by contact via the tarsal gustatory chemosensilla. The symptoms of their toxicity to insects and the rapid knockdown effect suggest neurotoxic action, but the precise mode of action and the molecular targets for decaleside action are not known. We have presented experimental evidence for the involvement of sodium pump inhibition in the insecticidal action of decaleside in the cockroach and housefly. The knockdown effect of decaleside is concomitant with the in vivo inhibition of Na+, K+ -ATPase in the head and thorax. The lack of insecticidal action by experimental ablation of tarsi or blocking the tarsal sites with paraffin correlated with lack of inhibition of Na+- K+ ATPase in vivo. Maltotriose, a trisaccharide, partially rescued the toxic action of decaleside as well as inhibition of the enzyme, suggesting the possible involvement of gustatory sugar receptors. In vitro studies with crude insect enzyme preparation and purified porcine Na+, K+ -ATPase showed that decaleside competitively inhibited the enzyme involving the ATP binding site. Our study shows that the insecticidal action of decaleside via the tarsal gustatory sites is causally linked to the inhibition of sodium pump which represents a unique mode of action. The precise target(s) for decaleside in the tarsal chemosensilla and the pathway linked to inhibition of sodium pump and the insecticidal action remain to be understood.

Molecular and functional characterization of a candidate sex pheromone receptor OR1 in Spodoptera litura.

Olfaction is primarily mediated by highly specified olfactory receptors (ORs). Here, we cloned and identified an olfactory receptor, named SlituOR1 (Genbank no. JN835269), from Spodoptera litura and found evidence that it is a candidate pheromone receptor. It exhibited male-biased expression in the antennae, where it was localized at the base of sensilla trichoidea, the antennal sensilla mainly responsive to pheromones in moths. Conserved orthologues of this receptor, found among known pheromone receptors within the Lepidoptera, and SlituOR1 were placed among a clade of candidate pheromone receptors in a phylogeny tree of insect OR gene sequences. SlituOR1 showed differential expression in S. litura populations attracted to traps baited with different ratios of the two sex pheromone components (9Z,11E)-tetradecadienyl acetate (Z9E11-14:OAc) and (9Z,12E)-tetradecadienyl acetate (Z9E12-14:OAc). Knocking down of SlituOR1 by RNA interference reduced the electroantennogram (EAG) response to Z9E11-14:OAc, and this result is consistent with the field trapping experiment. We infer that variation in transcription levels of olfactory receptors may modulate sex pheromone perception in male moths and could provide some of the flexibility required to maintain the functionality of communication with females when a population is adapting to a new niche and reproductive isolation becomes an advantage.

Trans-generational desensitization and within-generational resensitization of a sucrose-best neuron in the polyphagous herbivore Helicoverpa armigera (Lepidoptera: Noctuidae).

Dietary exposure of insects to a feeding deterrent substance for hours to days can induce habituation and concomitant desensitization of the response of peripheral gustatory neurons to such a substance. In the present study, larvae of the herbivore Helicoverpa armigera were fed on diets containing either a high, medium or low concentration of sucrose, a major feeding stimulant. The responsiveness of the sucrose-best neuron in the lateral sensilla styloconica on the galea was quantified. Results showed the response of the sucrose-best neuron exposed to high-sucrose diets decreased gradually over successive generations, resulting in complete desensitization in the 5th and subsequent generations. However, the sensitivity was completely restored in the ninth generation after neonate larvae were exposed to low-sucrose diet. These findings demonstrate phenotypic plasticity and exclude inadvertent artificial selection for low sensitivity to sucrose. No significant changes were found in the sensitivity of caterpillars which experienced low- or medium-sucrose diets over the same generations. Such desensitization versus re-sensitization did not generalise to the phagosimulant myo-inositol-sensitive neuron or the feeding deterrent-sensitive neuron. Our results demonstrate that under conditions of high sucrose availability trans-generational desensitization of a neuron sensitive to this feeding stimulant becomes more pronounced whereas re-sensitization occurs within one generation.

Larval sensilla of the moth Heliothis virescens respond to sex pheromone components.

Female-released sex pheromones orchestrate the mating behaviour of moths. Recent studies have shown that sex pheromones not only attract adult males but also caterpillars. Single sensillum recordings revealed that larval antennal sensilla of the moth Heliothis virescens respond to specific sex pheromone components. In search for the molecular basis of pheromone detection in larvae, we found that olfactory sensilla on the larval antennae are equipped with the same molecular elements that mediate sex pheromone detection in adult male moths, including the Heliothis virescens receptors 6 (HR6) and HR13, as well as sensory neurone membrane protein 1 (SNMP1). Thirty-eight olfactory sensory neurones were identified in three large sensilla basiconica; six of these are considered as candidate pheromone responsive cells based on the expression of SNMP1. The pheromone receptor HR6 was found to be expressed in two cells and the receptor HR13 in three cells. These putative pheromone responsive neurones were accompanied by cells expressing pheromone-binding protein 1 (PBP1) and PBP2. The results indicate that the responsiveness of larval sensilla to female-emitted sex pheromones is based on the same molecular machinery as in the antennae of adult males.

Smoke, pheromone and kairomone olfactory receptor neurons in males and females of the pine sawyer Monochamus galloprovincialis (Olivier) (Coleoptera: Cerambycidae).

The response of antennal olfactory receptor neurons (ORNs) of Monochamus galloprovincialis to several odourants was tested using single sensillum electrophysiology. Behaviourally active pheromone, and kairomone (host and sympatric bark beetle pheromone) odours were tested alongside smoke compounds released by burnt wood that are potentially attractive to the insect. The antennae bore several types of sensilla. Two plate areas in the proximal and distal ends of each antennal segment were covered with basiconic sensilla that responded to the odour stimuli. Sensilla basiconica contained one or two cells of different spike amplitude. The 32 male and 38 female ORNs tested responded with excitations or inhibitions to the different plant odours. In general the response of male and female receptors was very similar so they were pooled to perform a cluster analysis on ORN responses. Six ORNs were clearly specialised for pheromone reception. Responses to kairomone and smoke odours were less specific than those of pheromone, but a group of 9 cells was clearly excited by smoke compounds (mainly eugenol and 4-methyl 2-methoxyphenol), a group of 8 cells was very responsive to α-pinene, ß-pinene and cis-verbenol, and a group of 14 cells responded to a wider range of compounds. The rest of the cells (47%) were either non-responsive or slightly inhibited by smoke compounds. Dose-response curves were obtained for several compounds. Different compounds induced significantly different latencies and these appeared to be unrelated to their boiling point.

Gustatory sensitivity and food acceptance in two phylogenetically closely related papilionid species: Papilio hospiton and Papilio machaon.

In herbivorous insects, food selection depends on sensitivity to specific chemical stimuli from host-plants as well as to secondary metabolites (bitter) and to sugars (phagostimulatory). Bitter compounds are noxious, unpalatable or both and evoke an aversive feeding response. Instead, sugars and sugar alcohols play a critical role in determining and enhancing the palatability of foods. We assumed that peripheral taste sensitivity may be related to the width of the host selection. Our model consists of two closely phylogenetically related Papilionid species exhibiting a difference in host plant choice: Papilio hospiton and Papilio machaon. The spike activity of the lateral and medial maxillary styloconic taste sensilla was recorded following stimulation with several carbohydrates, nicotine and NaCl, with the aim of characterizing their gustatory receptor neurons and of comparing their response patterns in the light of their different acceptability in feeding behaviour. The results show that: a) each sensillum houses phagostimulant and phagodeterrent cells; b) the spike activity of the gustatory neurons in response to different taste stimuli is higher in P. hospiton than in P. machaon; c) sugar solutions inhibit the spike activity of the deterrent and salt cells, and the suppression is higher in P. machaon than in P. hospiton. In conclusion, we propose that the different balance between the phagostimulant and phagodeterrent inputs from GRNs of maxillary sensilla may contribute in determining the difference in food choice and host range.

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.

Olfactory responses of the antennal trichoid sensilla to chemical repellents in the mosquito, Culex quinquefasciatus.

Insect repellents are widely used to protect against insect bites and thus prevent allergic reaction and the spread of disease. To gain insight into the mosquito's response to chemicals repellents, we investigated the interaction between the olfactory system of the mosquito Culex quinquefasciatus Say and chemical repellents using single sensillum recording. The interactions of 50 repellent chemicals with olfactory receptor neurons were measured in six different types of mosquito sensilla: long sharp trichoid (LST), short sharp trichoid (SST), short blunt trichoid I (SBT-I), short blunt trichoid II (SBT-II), short blunt trichoid-curved (SBT-C), and grooved peg (GP). A single olfactory neuron reacted to the chemical repellents in each of the sensilla except for SBT-I and SBT-II, where two neurons were involved. Other than LST and GP, which showed no or very weak responses to the repellents tested, all the sensilla showed significant excitatory responses to certain types of repellents. Terpene-derived chemicals such as eucalyptol, α-pinene, and camphor, stimulated olfactory receptor neurons in a dose-dependent manner and mosquitoes responded more strongly to terpene-derived chemical repellents than to non-terpene-derived chemicals such as dimethyl phthalate. Mosquitoes also exhibited a similar response to stereoisomers of chemicals such as (-)-ß-pinene versus (+)-ß-pinene, and (-)-menthone versus (+)-menthone. This study not only demonstrates the effects of chemical repellents on the mosquito olfactory system but also provides important information that will assist those screening new mosquito repellents and designing new mosquito control agents.

An odorant-binding protein required for suppression of sweet taste by bitter chemicals.

Animals often must decide whether or not to consume a diet that contains competing attractive and aversive compounds. Here, using the fruit fly, Drosophila melanogaster, we describe a mechanism that influences this decision. Addition of bitter compounds to sucrose suppressed feeding behavior, and this inhibition depended on an odorant-binding protein (OBP) termed OBP49a. In wild-type flies, bitter compounds suppressed sucrose-induced action potentials, and the inhibition was impaired in Obp49a mutants. However, loss of OBP49a did not affect action potentials in sugar- or bitter-activated gustatory receptor neurons (GRNs) when the GRNs were presented with just one type of tastant. OBP49a was expressed in accessory cells and acted non-cell-autonomously to attenuate nerve firings in sugar-activated GRNs when bitter compounds were combined with sucrose. These findings demonstrate an unexpected role for an OBP in taste and identify a molecular player involved in the integration of opposing attractive and aversive gustatory inputs.

Acid sensing by sweet and bitter taste neurons in Drosophila melanogaster.

Drosophila melanogaster can taste various compounds and separate them into few basic categories such as sweet, bitter and salt taste. Here we investigate mechanisms underlying acid detection in Drosophila and report that the fly displays strong taste aversion to common carboxylic acids. We find that acid tastants act by the activation of a subset of bitter neurons and inhibition of sweet neurons. Bitter neurons begin to respond at pH 5 and show an increase in spike frequency as the extracellular pH drops, which does not rely on previously identified chemoreceptors. Notably, sweet neuron activity depends on the balance of sugar and acid tastant concentrations. This is independent of bitter neuron firing, and allows the fly to avoid acid-laced food sources even in the absence of functional bitter neurons. The two mechanisms may allow the fly to better evaluate the risk of ingesting acidic foods and modulate its feeding decisions accordingly.

Function and central projections of gustatory receptor neurons on the antenna of the noctuid moth Spodoptera littoralis.

Chemosensory information is crucial for most insects to feed and reproduce. Olfactory signals are mainly used at a distance, whereas gustatory stimuli play an important role when insects directly contact chemical substrates. In noctuid moths, although the antennae are the main olfactory organ, they also bear taste sensilla. These taste sensilla detect sugars and hence are involved in appetitive learning but could also play an important role in food evaluation by detecting salts and bitter substances. To investigate this, we measured the responses of individual taste sensilla on the antennae of Spodoptera littoralis to sugars and salts using tip recordings. We also traced the projections of their neuronal axons into the brain. In each sensillum, we found one or two neurons responding to sugars: one NaCl-responsive and one water-sensitive neuron. Responses of these neurons were dose-dependent and similar across different locations on the antenna. Responses were dependent on the sex for sucrose and on both sex and location for glucose and fructose. We did not observe a spatial map for the projections from specific regions of the antennae to the deutocerebrum or the tritocerebrum/suboesophageal ganglion complex. In accordance with physiological recordings, back-fills from individual sensilla revealed up to four axons, in most cases targeting different projection zones.