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Determining the molecular properties of neurons is essential to understand their development, function and evolution. Using Targeted DamID (TaDa), we characterize RNA polymerase II occupancy and chromatin accessibility in selected Ionotropic receptor ( Ir )-expressing olfactory sensory neurons in Drosophila . Although individual populations represent a minute fraction of cells, TaDa is sufficiently sensitive and specific to identify the expected receptor genes. Unique Ir expression is not consistently associated with differences in chromatin accessibility, but rather to distinct transcription factor profiles. Genes that are heterogeneously expressed across populations are enriched for neurodevelopmental factors, and we identify functions for the POU-domain protein Pdm3 as a genetic switch of Ir neuron fate, and the atypical cadherin Flamingo in segregation of neurons into discrete glomeruli. Together this study reveals the effectiveness of TaDa in profiling rare neural populations, identifies new roles for a transcription factor and a neuronal guidance molecule, and provides valuable datasets for future exploration., Competing Interests: JA, LA, JA, KM, PC, RB No competing interests declared, (© 2021, Arguello et al.)

Many animals use the Earth's geomagnetic field for orientation and navigation. Yet, the molecular and cellular underpinnings of the magnetic sense remain largely unknown. A biophysical model proposed that magnetoreception can be achieved through quantum effects of magnetically-sensitive radical pairs formed by the photoexcitation of cryptochrome (CRY) proteins. Studies in Drosophila are the only ones to date to have provided compelling evidence for the ultraviolet (UV)-A/blue light-sensitive type 1 CRY (CRY1) involvement in animal magnetoreception, and surprisingly extended this discovery to the light-insensitive mammalian-like type 2 CRYs (CRY2s) of both monarchs and humans. Here, we show that monarchs respond to a reversal of the inclination of the Earth's magnetic field in an UV-A/blue light and CRY1, but not CRY2, dependent manner. We further demonstrate that both antennae and eyes, which express CRY1, are magnetosensory organs. Our work argues that only light-sensitive CRYs function in animal light-dependent inclination-based magnetic sensing.

Reproductive division of labor in insect societies is regulated through multiple concurrent mechanisms, primarily chemical and behavioral. Here, we examined if the Dufour's gland secretion in the primitively eusocial bumble bee Bombus impatiens signals information about caste, social condition, and reproductive status. We chemically analyzed Dufour's gland contents across castes, age groups, social and reproductive conditions, and examined worker behavioral and antennal responses to gland extracts. We found that workers and queens each possess caste-specific compounds in their Dufour's glands. Queens and gynes differed from workers based on the presence of diterpene compounds which were absent in workers, whereas four esters were exclusive to workers. These esters, as well as the total amounts of hydrocarbons in the gland, provided a separation between castes and also between fertile and sterile workers. Olfactometer bioassays demonstrated attraction of workers to Dufour's gland extracts that did not represent a reproductive conflict, while electroantennogram recordings showed higher overall antennal sensitivity in queenless workers. Our results demonstrate that compounds in the Dufour's gland act as caste- and physiology-specific signals and are used by workers to discriminate between workers of different social and reproductive status.

The Southern house mosquito Culex quinquefasciatus relies on its olfactory system to locate the human hosts for blood meals, by which several deadly diseases are transmitted. Olfactory sensory neurons (OSNs) housed in the sensilla on the olfactory appendages send their axons into the antennal lobes (ALs), the primary olfactory center in the brain, where the OSNs expressing the same olfactory receptors converge upon the same spherical structures known as glomeruli in the AL. The structure of the antennal lobe, that is, the spatial organization of the glomeruli, governs the insect's odor identification and discrimination. Drosophila studies have demonstrated the specific connections between receptors and glomeruli based on the 3D structure of the antennal lobe, deepening our understanding of the relationships between glomerular activities and behaviors, but as yet the structure of the Cx. quinquefasciatus antennal lobe remains unknown. We therefore constructed a 3D model of the Cx. quinquefasciatus antennal lobe using nc82 antibody staining, identifying 62 and 44 glomeruli in the female and male mosquito antennal lobe, respectively, with a significant sexual dimorphism in terms of the antennal lobe volume and glomerulus number. These results demonstrate the structural basis of mosquito odor coding and provide a platform for future studies of the mosquito olfactory signal processing mechanism., (© 2020 Institute of Zoology, Chinese Academy of Sciences.)

The use of Erianthus arundinaceus as a trap plant in association with sugarcane reduces populations of the spotted sugarcane stalk borer Chilo sacchariphagus (Bojer) (Lepidoptera: Crambidae). This grass acts as a dead-end trap crop because it is the preferred plant for oviposition relative to sugarcane, and it precludes larval development. We explored the chemical mechanisms involved in host choice by C. sacchariphagus. We showed that the insect's antennal receptors are particularly sensitive to the shared compounds found in the volatile emissions produced by sugarcane and E. arundinaceus. In accordance with their phylogenetic proximity, the two plant species share many physicochemical properties, which suggests that C. sacchariphagus has few sensory cues to differentiate between the two. The terpene (E)-β-ocimene is constitutively emitted by E. arundinaceus, but not by sugarcane. It elicits an electroantennographic response and behavioral responses from female C. sacchariphagus in Y-tube bioassays. Our study suggests that the sensory confusion between host plants, combined with a marginal sensory difference orienting the choice of an egg-laying site, constitutes a mechanism that is relevant to trap cropping. Systems based on this type of mechanism could provide long-term protection for crops vulnerable to insect pests.

The study of insect semiochemicals, especially pheromones, is of fundamental importance for the development of strategies for controlling agricultural pests. In this study, volatile compounds involved in the communication between males and females of the fruit fly, Anastrepha obliqua (Diptera: Tephritidae), for mating purposes were characterized to develop attractant formulations for females of this species. Extracts containing volatile compounds released by males of A. obliqua were obtained by the dynamic headspace technique and analyzed by gas chromatography coupled with an electroantennographic detector (GC-EAD) and gas chromatography coupled with mass spectrometry (GC-MS). Twenty-one volatile compounds were identified in the aeration extracts of males. Five of them caused EAD responses from the antennae of females: 1-heptanol, linalool, (Z)-3-nonen-1-ol, (E,Z)-3,6-nonadien-1-ol, and (Z,E)-α-farnesene. Six synthetic mixtures of these compounds, including the five-component blend and all possible four-component blends, were formulated in a biopolymer and used in behavioral bioassays conducted in the laboratory arena with conspecific virgin females. One blend of 1-heptanol, linalool, (Z)-3-nonen-1-ol, and (Z,E)-α-farnesene attracted more females than the collection of volatiles from virgin males used as control. The other mixtures were as attractive to A. obliqua females as the control treatment. This study indicates potential for use of these compounds in monitoring and control strategies for this pest.

Research on the functions of insect chemoreceptors have primarily focused on antennae (olfactory receptors) and mouthparts (gustatory receptors). However, chemoreceptive sensilla are also present on other appendages, such as the leg tarsi and the anterior wing margin, and their specific roles in chemoreception and mosquito behavior remain largely unknown. In this study, electrophysiological analyses in an electroantennogram recording format were performed on Aedes aegypti (L., Diptera: Culicidae) antennae, mouthparts, tarsi, and wings during exposure to a variety of insect repellent and attractant compounds. The results provide evidence that the tarsi and wings can sense chemicals in a gaseous form, and that the odors produce differing responses on different appendages. The most consistent and strongest response occurred when exposed to triethylamine (TEA). Antennae and mouthparts showed nearly identical responses pattern to all tested compounds, and their rank orders of effectiveness were similar to those of fore- and mid-leg tarsi. Hindleg tarsi only responded to TEA, indicating that the hind legs are not as chemoreceptive. Wings responded to a range of odorants, but with a different rank order and voltage amplitude. Insights gleaned into the function of these appendages in insect chemoreception are discussed., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Recently, insect-machine hybrid robots have been developed that incorporate insects into robots or incorporate machines into insects. Most previous studies were motivated to use the function of insects for robots, but this technology can also prove to be useful as an experimental tool for neuroethology. We reviewed hybrid robots in terms of the closed-loop between an insect, a robot, and the real environment. The incorporated biological components provided the robot sensory signals that were received by the insects and the adaptive functions of the brain. The incorporated artificial components permitted us to understand the biological system by controlling insect behavior. Hybrid robots thus extend the roles of mobile robot experiments in neuroethology for both model evaluation and brain function analysis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Flying insects are known to orient themselves over large distances using minute amounts of odors. Some bear pectinate antennae of remarkable architecture thought to improve olfactory performance. The semiporous, multiscale nature of these antennae influences how odor molecules reach their surface. We focus here on the repeating structural building blocks of these antennae in Saturniid moths. This microstructure consists of one ramus or branch and its many hair-like sensilla, responsible for chemical detection. We experimentally determined leakiness, defined as the proportion of air going through the microstructure rather than flowing around it, by particle image velocimetry visualization of the flow around three-dimensional printed scaled-up mock-ups. The combination of these results with a model of mass transfer showed that most pheromone molecules are deflected around the microstructure at low flow velocities, keeping them out of reach. Capture is thus determined by leakiness. By contrast, at high velocities, molecular diffusion is too slow to be effective, and the molecules pass through the structure without being captured. The sensory structure displays maximal odor capture efficiency at intermediate flow speeds, as encountered by the animal during flight. These findings also provide a rationale for the previously described "olfactory lens," an increase in pheromone reception at the proximal end of the sensors. We posit that it is based on passive mass transfer rather than on physicochemical surface processes., Competing Interests: The authors declare no competing interest.

Allotraeus asiaticus Schwarzer and Callidiellum villosulum Fairmaire are repeatedly intercepted in wood and wood products all over the world. As two common stem borers of Cunninghamia lanceolata (Lambert) Hooker, to further understanding of the differences in their living habits, behaviors and the mechanism of insect-host chemical communication, we observed the external morphology, number and distribution of antennal sensilla of A. asiaticus and C. villosulum with scanning electron microscopy (SEM), respectively. The results showed that 1st-5th subsegments of the flagellum are spined endoapically in A. asiaticus which is different from the previous report (1st-3rd of the flagellomere). Meanwhile, there were five subsegments on the flagellum of C. villosulum that were clearly specialized as serrated shapes on the 4th-8th flagellomeres. Four types (ten subtypes) of sensilla were both found on the antennae of these two fir longhorn beetles, named Böhm bristle (Bb), sensilla trichodea (ST I and II), sensilla basiconica (SB I, II and III), sensilla chaetica (SCh I, II, III and IV). There is one additional kind of morphological type of sensilla found on the antennae of C. villosulum compared to A. asiaticus which was related to their habit of laying eggs only on dry and injured fir branches, named sensilla campaniformia (SCa). These differences may vary according to their own biological habits. For research purposes, the observed difference in the sensillum distribution and function between the two fir longhorn beetles will greatly facilitate the design of better semiochemical control methods of these insect pests., Competing Interests: The authors have declared that no competing interests exist.

Diverse mechanosensory neurons detect different mechanical forces that can impact animal behavior. Yet our understanding of the anatomical and physiological diversity of these neurons and the behaviors that they influence is limited. We previously discovered that grooming of the Drosophila melanogaster antennae is elicited by an antennal mechanosensory chordotonal organ, the Johnston's organ (JO) (Hampel et al., 2015). Here, we describe anatomically and physiologically distinct JO mechanosensory neuron subpopulations that each elicit antennal grooming. We show that the subpopulations project to different, discrete zones in the brain and differ in their responses to mechanical stimulation of the antennae. Although activation of each subpopulation elicits antennal grooming, distinct subpopulations also elicit the additional behaviors of wing flapping or backward locomotion. Our results provide a comprehensive description of the diversity of mechanosensory neurons in the JO, and reveal that distinct JO subpopulations can elicit both common and distinct behavioral responses., Competing Interests: SH, KE, DY, DB, AK, AS No competing interests declared, (© 2020, Hampel et al.)

The yellow fever mosquito Aedes aegypti is a prolific vector of arboviral and filarial diseases that largely relies on its sense of smell to find humans. To facilitate in-depth analysis of the neural circuitry underlying Ae. aegypti olfactory-driven behaviors, we generated an updated in vitro atlas for the antennal lobe olfactory brain region of this disease vector using two independent neuronal staining methods. We performed morphological reconstructions with replicate fixed, dissected and stained brain samples from adult male and female Ae. aegypti of the LVPib12 genome reference strain and determined that the antennal lobe in both sexes is comprised of approximately 80 discrete glomeruli. Guided by landmark features in the antennal lobe, we found 63 of these glomeruli are stereotypically located in spatially invariant positions within these in vitro preparations. A posteriorly positioned, mediodorsal glomerulus denoted MD1 was identified as the largest spatially invariant glomerulus in the antennal lobe. Spatial organization of glomeruli in a recently field-derived strain of Ae. aegypti from Puerto Rico was conserved, despite differences in antennal lobe shape relative to the inbred LVPib12 strain. This model in vitro atlas will serve as a useful community resource to improve antennal lobe annotation and anatomically map projection patterns of neurons expressing target genes in this olfactory center. It will also facilitate the development of chemotopic maps of odor representation in the mosquito antennal lobe to decode the molecular and cellular basis of Ae. aegypti attraction to human scent and other chemosensory cues., Competing Interests: The authors have declared that no competing interests exist.

Amecephala pusilla gen. et sp. nov. is described and illustrated on the basis of a well-preserved female psyllid (Liadopsyllidae) in a piece of Cretaceous Myanmar amber. The new genus differs from other members of Liadopsyllidae in details of the antennae and forewings. For the first time, the presence of a circumanal ring is documented for Mesozoic psyllids. Based on differences in the length of female terminalia, it is suggested that Liadopsyllidae may have displayed a diversified oviposition biology. As far as known, Liadopsyllidae lack a pulvillus, a putative autapomorphy supporting the monophyly of Liadopsyllidae. An identification key to genera and an annotated checklist of known Liadopsyllidae species are provided. New synonyms and combinations are proposed and the status of the subfamily Miralinae is discussed.

Moths often use multi-component pheromones with fixed ratios to keep intraspecific communication and interspecific isolation. Unusually, the Oriental armyworm Mythimna separata in North China use only Z11-16:Ald as the essential component of its sex pheromone to find mates. To understand how this species keeps behavioral isolation from other species sharing Z11-16:Ald as a major pheromone component, we study the olfactory coding of intra- and interspecific pheromonal messages in the males of M. separata. Firstly, we functionally characterized the long trichoid sensilla in male antennae by single sensillum recording. Two types of sensilla were classified: the A type sensilla responded to Z11-16:Ald and Z9-14:Ald, and the B type sensilla mainly to Z9-14:Ald, and also to Z11-16:Ac, Z11-16:OH, and Z9-16:Ald. Next, we examined the glomerulus responses in the antennal lobes to these compounds by using in vivo optical imaging. The results showed that among the three subunits of the macroglomerular complex (MGC), Z11-16:Ald activated the cumulus, Z9-14:Ald activated the dorso-anterior and the cumulus, Z11-16:OH and Z11-16:Ac activated the dorso-anterior and dorso-posterior, respectively. However, Z9-16:Ald activated an ordinary glomerulus. Thirdly, we tested the behavioral responses of the males to these compounds in the wind tunnel. Addition of Z9-14:Ald at the ratio of 1:10 greatly reduced the attractiveness of Z11-16:Ald, addition of Z9-16:Ald or Z11-16:OH at the ratio of 1:1 also had behavioral antagonistic effects, while addition of Z11-16:Ac had no effect on the attractiveness of Z11-16:Ald. Finally, we used antennal transcriptome data and the Xenopus expression system to identify the receptor of Z9-14:Ald in M. separata. The Xenopus oocytes co-expressing MsepOR2 and MsepORco showed a strong response to Z9-14:Ald. Two-color fluorescence in situ hybridization validated that the cells expressing MsepOR2 and MsepOR3, tuned to Z9-14:Ald and Z11-16:Ald respectively, were localized in the different sensilla of male antennae. Comparing the sex pheromone communication channel of the related species, our results suggest that the conserved olfactory pathways for behavioral antagonists play a crucial role in behavioral isolation of noctuid species., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Gills and the antennal gland are ion-regulatory organs in crabs. Previous studies have suggested that the differences in the morphology and ion regulation of gills and accessory respiratory organs between ocypodid and grapsid species are related to their distinct evolutionary transition to land habitats. In addition, Na + , K + -ATPase (NKA) activity and Na + and NH 4 + regulation in the antennal gland differ between ocypodid and grapsid species, which had different terrestrial adaptation trajectories. This study used five Ocypodoidea species and three Grapsoidea species from the intertidal and supratidal zones to further investigate the differences in ion regulation and NKA activity in the antennal gland between these crab families in different habitats. Crabs were transferred to 5 practical salinity unit (PSU) water, and osmolality, Na + and Cl - concentrations in the urine and hemolymph, and NKA activity in the antennal gland were examined. Phylogenetic ANOVA results showed that the NKA activity in the antennal gland was higher in the ocypodid than grapsid groups, and Moran's I autocorrelation analysis also indicated that NKA activity in the antennal gland was phylogenetically correlated among crabs. K-means clustering showed a difference among the crabs in the crabs' Na + and Cl - concentrations in the urine/hemolymph, NKA activities in the antennal gland and gill 6, and number of pairs of gills. Crabs with relatively high antennal gland NKA activity were found not only in the Ocypode species, which are better adapted to terrestrial environments, but also in two intertidal species of Gelasiminae. In conclusion, part of the Ocypodidae lineage may have a) the ability to reabsorb Na + and b) higher NKA activity in the antennal gland than other families, and this phenomenon is phylogenetically correlated in Ocypodoidea and Grapsoidea. The physiological diversity in osmoregulation among intertidal and costal species provides a base to further investigate their ecological niches and guilds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pathogens may manipulate their human and mosquito hosts to enhance disease transmission. Dengue, caused by four viral serotypes, is the fastest-growing transmissible disease globally resulting in 50-100 million infections annually. Transmission of the disease relies on the interaction between humans and the vector Aedes aegypti and is largely dependent on the odor-mediated host seeking of female mosquitoes. In this study, we use activity monitors to demonstrate that dengue virus-1 affects the locomotion and odor-mediated behavior of Ae. aegypti, reflecting the progression of infection within the mosquito. Mosquitoes 4-6 days post-infection increase locomotion, but do not alter their odor-driven host-seeking response. In contrast, females 14-16 days post-infection are less active, yet more sensitive to human odors as assessed by behavioral and electrophysiological assays. Such an increase in physiological and behavioral sensitivity is reflected by the antennal-specific increase in abundance of neural signaling transcripts in 14 days post-infection females, as determined by transcriptome analysis. This suggests that the sensitivity of the mosquito peripheral olfactory system is altered by the dengue virus by enhancing the overall neural responsiveness of the antenna, rather than the selective regulation of chemosensory-related genes. Our study reveals that dengue virus-1 enhances vector-related behaviors in the early stages post-infection that aid in avoiding predation and increasing spatial exploration. On the other hand, at the later stages of infection, the virus enhances the host-seeking capacity of the vector, thereby increasing the risk of virus transmission. A potential mechanism is discussed., Competing Interests: The authors have declared that no competing interests exist.

Spatial maps in the brain are most accurate when they are linked to external sensory cues. Here, we show that the compass in the Drosophila brain is linked to the direction of the wind. Shifting the wind rightward rotates the compass as if the fly were turning leftward, and vice versa. We describe the mechanisms of several computations that integrate wind information into the compass. First, an intensity-invariant representation of wind direction is computed by comparing left-right mechanosensory signals. Then, signals are reformatted to reduce the coding biases inherent in peripheral mechanics, and wind cues are brought into the same circular coordinate system that represents visual cues and self-motion signals. Because the compass incorporates both mechanosensory and visual cues, it should enable navigation under conditions where no single cue is consistently reliable. These results show how local sensory signals can be transformed into a global, multimodal, abstract representation of space., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The lady beetle Hippodamia variegata is an important biocontrol agent of many aphids. In this study, the fine morphology of antennae as well as the typology, morphology and distribution of antennal sensilla were comprehensively examined by scanning electron microscopy. The antennal morphology of female and male are similar and consist of the scape, pedicel, and nine flagellomeres. No significant difference was detected in the length of each segment between two sexes, while the male antennae are much stronger than females. In total, six types of sensilla can be defined on antenna, including Böhm bristle, sensilla chaetica (with three subtypes), sensilla basiconica (with three subtypes), sensilla trichodea, sensilla placodea and sensilla coeloconica. It is worth noting that sensilla chaetica III distributed only on the fixed position of male antennae. In addition, the functional morphology of antennae of H. variegata were compared with other lady beetles from multiple perspectives. Specially, the function of sensilla were also discussed according to their morphology, location and information from previous studies., Competing Interests: The authors have declared that no competing interests exist.

Calcium is an important intracellular second messenger involved in several processes such as the transduction of odour signals and neuronal excitability. Despite this critical role, relatively little information is available with respect to the impact of insecticides on the dynamics of intracellular calcium homeostasis in olfactory neurons. For the first time here, physiological stimuli (depolarizing current or pheromone) were shown to elicit calcium transients in peripheral neurons from the honey bee antenna. In addition, neurotoxic xenobiotics (the first synthetic phthalic diamide insecticide flubendiamide or botanical alkaloids ryanodine and caffeine) do interfere with normal calcium homeostasis. Our in vitro experiments show that these three xenobiotics can induce sustained abnormal calcium transients in antennal neurons. The present results provide a new insight into the toxicity of diamides, showing that flubendiamide drastically impairs calcium homeostasis in antennal neurons. We propose that a calcium imaging assay should provide an efficient tool dedicated to the modern assessment strategies of insecticides toxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Insect sensing of odorants plays important roles in various behaviors, including host location, mate attraction, and oviposition site selection. The odorant receptor (OR) is a key protein in insect environmental odor recognition. Most Diptera studies of ORs have focused on Drosophila and mosquitos, so there little known about ORs in the agricultural pest insects Tephritidae. To understand the olfactory recognition mechanism of Bactrocera minax, we sequenced and analyzed 12 B. minax transcriptomes to identify a total of 59 OR genes. Semi-quantitative reverse transcription PCR (RT-PCR) showed that several BminORs were highly expressed in antennae. Available with a complete open reading frame and expressed in the antennae of both sexes at a higher level than those of other BminORs, BminOR24 was selected for further functional analyses. BminOR24/BminOrco expressed in Xenopus oocytes responded significantly to linalool. The identification of B. minax OR genes lays a foundation for further functional studies of OR genes, and functional characterization of BminOR24 provides insight for improving methods for controlling B. minax, a devastating pest insects., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Portable electroantennograms (pEAG) can further our understanding of odor plume dynamics and complement laboratory-based electroantennogram tools. pEAG's can help to address important questions such as the influence of plume structure on insect behavior, the active space of semiochemical-baited traps, and the impact of biotic and abiotic factors on this active space. Challenges associated with pEAGs include their miniaturization and sensitivity, confounding environmental odors, and processing of data. Here, we describe a pEAG built with modern engineering hardware and techniques that is portable in being both light in weight (516 g) and smaller (12 × 12 × 8 cm, volume 1152 cm 3 ) than earlier models. It is able to incorporate insects of a range of sizes (4 to 30 mm antennal length), has wireless communication (communication range of 600 m urban, 10 km line of sight), a stand-alone power supply, and uses both antennae of the test insect. We report normalized antennal responses from Epiphyas postvittana in a dose response experiment where our pEAG compared favorably with traditional laboratory EAG equipment for this species. Dose-response comparisons between E. postvittana, Agrotis ipsilon, and Lymantria dispar dispar showed mean detection limits from a pheromone source dose of 100, 100, and 1 ng, respectively, for our pEAG. This pEAG should allow future real-time analysis of EAG responses in the field in research on how insects interact with odor plumes and the factors that influence the active space of semiochemical-baited traps.

The examination of phylogenetic and phenotypic characteristics of the nervous system, such as behavior and neuroanatomy, can be utilized as a means to assess speciation. Recent studies have proposed a fundamental tradeoff between two sensory organs, the eye and the antenna. However, the identification of ecological mechanisms for this observed tradeoff have not been firmly established. Our current study examines several monophyletic species within the obscura group, and asserts that despite their close relatedness and overlapping ecology, they deviate strongly in both visual and olfactory investment. We contend that both courtship and microhabitat preferences support the observed inverse variation in these sensory traits. Here, this variation in visual and olfactory investment seems to provide relaxed competition, a process by which similar species can use a shared environment differently and in ways that help them coexist. Moreover, that behavioral separation according to light gradients occurs first, and subsequently, courtship deviations arise., Competing Interests: IK, VG, MK, BH No competing interests declared, (© 2020, Keesey et al.)

Diaphorina citri Kuwayama is the most economically important citrus pest which is the primary vector of Candidatus Liberibacter spp. causing citrus greening (huanglongbing, HLB) disease. To better understand the developmental and structural changes of antennae and antennal sensilla in D. citri nymphs, we investigated the antennal morphology, structure and sensilla distribution of the five nymphal stages of D. citri using scanning electron microscopy. The antennae of the five different nymphal stages of D. citri were filiform in shape, which consisted of two segments in the first-, second- and third-instar nymphs; three segments in the fourth- and fifth-instar nymphs. The length of their antennae was significantly increased with the increase of the nymphal instar, as well as the total number of antennal sensilla. Ten morphological sensilla types were recorded altogether. They were the long terminal hair (TH1), short terminal hair (TH2), sensilla trichoidea (ST), cavity sensillum 1 (CvS1), cavity sensillum 2 (CvS2), sensilla basiconica 1-3 (SB1-3), sensilla campaniform (SCA) and partitioned sensory organ (PSO). Also, the distribution of antennal sensilla in each nymphal stage of D. citri was asymmetrical. The SBs only occurred on the antennae of the third-, fourth- and fifth-instar nymphs. Only one CvS2 was found in the third- and fifth-instar nymphs, and one SCA in the fourth- and fifth-instar nymphs, respectively. The possible roles of the nymphal antennal sensilla in D. citri were discussed. The results could contribute to a better understanding of the development of the sensory system, and facilitate future studies on the antennal functions in D. citri nymphs., Competing Interests: The authors have declared that no competing interests exist.

Some flying animals use active sensing to perceive and avoid obstacles. Nocturnal mosquitoes exhibit a behavioral response to divert away from surfaces when vision is unavailable, indicating a short-range, mechanosensory collision-avoidance mechanism. We suggest that this behavior is mediated by perceiving modulations of their self-induced airflow patterns as they enter a ground or wall effect. We used computational fluid dynamics simulations of low-altitude and near-wall flights based on in vivo high-speed kinematic measurements to quantify changes in the self-generated pressure and velocity cues at the sensitive mechanosensory antennae. We validated the principle that encoding aerodynamic information can enable collision avoidance by developing a quadcopter with a sensory system inspired by the mosquito. Such low-power sensing systems have major potential for future use in safer rotorcraft control systems., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Age-related hearing loss (ARHL) is a threat to future human wellbeing. Multiple factors contributing to the terminal auditory decline have been identified; but a unified understanding of ARHL - or the homeostatic maintenance of hearing before its breakdown - is missing. We here present an in-depth analysis of homeostasis and ageing in the antennal ears of the fruit fly Drosophila melanogaster. We show that Drosophila, just like humans, display ARHL. By focusing on the phase of dynamic stability prior to the eventual hearing loss we discovered a set of evolutionarily conserved homeostasis genes. The transcription factors Onecut (closest human orthologues: ONECUT2, ONECUT3), Optix (SIX3, SIX6), Worniu (SNAI2) and Amos (ATOH1, ATOH7, ATOH8, NEUROD1) emerged as key regulators, acting upstream of core components of the fly's molecular machinery for auditory transduction and amplification. Adult-specific manipulation of homeostatic regulators in the fly's auditory neurons accelerated - or protected against - ARHL.

House flies (Musca domestica L.) are mechanical vectors of food-borne pathogens including Salmonella spp., Escherichia coli O157:H7, and Shigella spp., resulting in increased risk of diarrheal disease in areas where flies are abundant. Movement of house flies into food crops may be increased by the presence of honeydew-producing insects feeding on these crops. Using gas chromatography-electroantennogram detection (GC-EAD) and gas chromatography-mass spectrometry (GC-MS), volatile odors that elicited house fly antennal response were identified from naval orange (Osbeck) (Sapindales: Rutaceae) and Marsh grapefruit (Macfad.) (Sapindales: Rutaceae) leaves infested with whitefly (Hemiptera: Aleyrodidae) and from whole faba (L.) (Fabales: Fabaceae) bean plants infested with aphids (Hemiptera: Aphididae). Volatiles identified included benzaldehyde, butyl hexanoate, β-caryophyllene, Δ3-carene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), (Z)-3-hexenyl acetate, myrcene, limonene, linalool, and naphthalene. This was followed by semifield bioassays of volatile blends and individual volatiles to determine house fly attraction to these volatiles. Although fly capture rates in the semifield setting were low, benzaldehyde and (Z)-3-hexenyl acetate were consistently attractive to house flies as individual compounds and as components of volatile blends., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

As the oldest, but least understood sensory system in evolution, the olfactory system represents one of the most challenging research targets in sensory neurobiology. Although a large number of computational models of the olfactory system have been proposed, they do not account for the diversity in physiology, connectivity of local neurons, and several recent discoveries in the insect antennal lobe, a major olfactory organ in insects. Recent studies revealed that the response of some projection neurons were reduced by application of a GABA antagonist, and that insects are sensitive to odor pulse frequency. To account for these observations, we propose a spiking neural circuit model of the insect antennal lobe. Based on recent anatomical and physiological studies, we included three sub-types of local neurons as well as synaptic short-term depression (STD) in the model and showed that the interaction between STD and local neurons resulted in frequency-sensitive responses. We further discovered that the unexpected response of the projection neurons to the GABA antagonist is the result of complex interactions between STD and presynaptic inhibition, which is required for enhancing sensitivity to odor stimuli. Finally, we found that odor discrimination is improved if the innervation of the local neurons in the glomeruli follows a specific pattern. Our findings suggest that STD, presynaptic inhibition and diverse physiology and connectivity of local neurons are not independent properties, but they interact to play key roles in the function of antennal lobes.

The giant sugarcane borer Telchin licus (Drury, 1773) (Lepidoptera: Castniidae) is a day-flying moth pest of sugarcane, pineapples and bananas. To better understand the chemical communication in this species, we examined the morphology of its olfactory system and the chemical composition of its body parts. The ventral surface of the clubbed antennae of T. licus has six morphological types of sensilla: sensilla trichodea, basiconica, chaetica, squamiforma, coeloconica, and auricillica. The telescopic ovipositor shows no evidence of a sexual gland, or female-specific compounds. On the other hand, the midleg basitarsus of males releases (E,Z)-2,13-octadecadienol and (Z,E)-2,13-octadecadienol, which are electroantennographically active in both sexes. These compounds are known female sex pheromones in the Sesiidae family and are male-specific compounds in another castniid moth, although further investigations are necessary to elucidate their ecological role in the Castniidae family., Competing Interests: The authors have declared that no competing interests exist.

Pyrethroids are synthetic insecticides that have a repellent action. This effect has been associated with an increase in the locomotor activity, which causes the avoidance of the insecticide-treated area (excito-repellency). In this work, we studied with behavior and electrophysiological recordings the occurrence of olfactory-mediated repellency caused by pyrethroids of different volatility in the German cockroach Blattella germanica (Linnaeus, 1767). Male cockroaches were spatially repelled when they were exposed to D-allethrin vapors and vapothrin vapors in a dose-dependent manner. No repellency was observed when insects were exposed to permethrin, a non-volatile pyrethroid. To confirm the role of olfaction in this phenomenon, we measured the electrical activity of the cockroaches' antennae in response to these insecticides. There was a significant increase in the electrical activity in response to D-allethrin and vapothrin, but no increase was observed in insects exposed to permethrin. Locomotor activity of cockroaches exposed to pyrethroids was measured in order to discard excito-repellency. No changes in locomotor activity were observed for any of the insecticides. Finally, we found that volatile pyrethroids in the vapor phase cause spatial repellency in cockroaches, being the first report of an olfactory-mediated repellency phenomenon caused by pyrethroids in cockroaches.

Serotonin (5-HT) represents a quintessential neuromodulator, having been identified in nearly all animal species [1] where it functions in cognition [2], motor control [3], and sensory processing [4]. In the olfactory circuits of flies and mice, serotonin indirectly inhibits odor responses in olfactory receptor neurons (ORNs) via GABAergic local interneurons (LNs) [5, 6]. However, the effects of 5-HT in olfaction are likely complicated, because multiple receptor subtypes are distributed throughout the olfactory bulb (OB) and antennal lobe (AL), the first layers of olfactory neuropil in mammals and insects, respectively [7]. For example, serotonin has a non-monotonic effect on odor responses in Drosophila projection neurons (PNs), where low concentrations suppress odor-evoked activity and higher concentrations boost PN responses [8]. Serotonin reaches the AL via the diffusion of paracrine 5-HT through the fly hemolymph [8] and by activation of the contralaterally projecting serotonin-immunoreactive deuterocerebral interneurons (CSDns): the only serotonergic cells that innervate the AL [9, 10]. Concentration-dependent effects could arise by either the expression of multiple 5-HT receptors (5-HTRs) on the same cells or by populations of neurons dedicated to detecting serotonin at different concentrations. Here, we identify a population of LNs that express 5-HT7Rs exclusively to detect basal concentrations of 5-HT. These LNs inhibit PNs via GABA B receptors and mediate subtractive gain control. LNs expressing 5-HT7Rs are broadly tuned to odors and target every glomerulus in the antennal lobe. Our results demonstrate that serotonergic modulation at low concentrations targets a specific population of LNs to globally downregulate PN odor responses in the AL., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bed bugs are pests of public health importance due to their relentless biting habits that can lead to allergies, secondary infections and mental health issues. When not feeding on human blood bed bugs aggregate in refuges close to human hosts. This aggregation behaviour could be exploited to lure bed bugs into traps for surveillance, treatment efficacy monitoring and mass trapping efforts, if the responsible cues are identified. The aim of this study was to identify and quantify the bed bug aggregation pheromone. Volatile chemicals were collected from bed bug-exposed papers, which are known to induce aggregation behaviour, by air entrainment. This extract was tested for behavioural and electrophysiological activity using a still-air olfactometer and electroantennography, respectively. Coupled gas chromatography-electroantennography (GC-EAG) was used to screen the extract and the GC-EAG-active chemicals, benzaldehyde, hexanal, (E)-2-octenal, octanal, nonanal, decanal, heptanal, (R,S)-1-octen-3-ol, 3-carene, β-phellandrene, (3E,5E)-octadien-2-one, (E)-2-nonenal, 2-decanone, dodecane, nonanoic acid, 2-(2-butoxyethoxy)ethyl acetate, (E)-2-undecanal and (S)-germacrene D, were identified by GC-mass spectrometry and quantified by GC. Synthetic blends, comprising 6, 16, and 18 compounds, at natural ratios, were then tested in the still-air olfactometer to determine behavioural activity. These aggregation chemicals can be manufactured into a lure that could be used to improve bed bug management.

Bactrocera frauenfeldi (Schiner) (Diptera: Tephritidae) is a polyphagous fruit fly pest species that is endemic to Papua New Guinea and has become established in several Pacific Islands and Australia. Despite its economic importance for many crops and the key role of chemical-mediated sexual communication in the reproductive biology of tephritid fruit flies, as well as the potential application of pheromones as attractants, there have been no studies investigating the identity or activity of rectal gland secretions or emission profiles of this species. The present study (1) identifies the chemical profile of volatile compounds produced in rectal glands and released by B. frauenfeldi , (2) investigates which of the volatile compounds elicit an electroantennographic or electropalpographic response, and (3) investigates the potential function of glandular emissions as mate-attracting sex pheromones. Rectal gland extracts and headspace collections from sexually mature males and females of B. frauenfeldi were analysed by gas chromatography-mass spectrometry. Male rectal glands contained ( E , E )-2-ethyl-8-methyl-1,7-dioxaspiro [5.5]undecane as a major component and ( E , E )-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane as a moderate component. Minor components included palmitoleic acid, palmitic acid, and ethyl oleate. In contrast, female rectal glands contained ( E , E )-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and ethyl laurate as major components, ethyl myristate and ethyl palmitoleate as moderate components, and 18 minor compounds including amides, esters, and spiroacetals. Although fewer compounds were detected from the headspace collections of both males and females than from the gland extractions, most of the abundant chemicals in the rectal gland extracts were also detected in the headspace collections. Gas chromatography coupled electroantennographic detection found responses to ( E , E )-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane from the antennae of both male and female B. frauenfeldi . Responses to ( E , E )-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane were elicited from the antennae of females but not males. The two spiroacetals also elicited electropalpographic responses from both male and female B. frauenfeldi . Ethyl caprate and methyl laurate, found in female rectal glands, elicited responses in female antennae and palps, respectively. Y-maze bioassays showed that females were attracted to the volatiles from male rectal glands but males were not. Neither males nor females were attracted to the volatiles from female rectal glands. Our findings suggest ( E , E )-2,8-dimethyl-1,7-dioxaspiro[5.5]undecane and ( E , E )-2-ethyl-8-methyl-1,7-dioxaspiro[5.5]undecane as components of a sex-attracting pheromone in B. frauenfeldi .

Connections between neuronal populations may be genetically hardwired or random. In the insect olfactory system, projection neurons of the antennal lobe connect randomly to Kenyon cells of the mushroom body. Consequently, while the odor responses of the projection neurons are stereotyped across individuals, the responses of the Kenyon cells are variable. Surprisingly, downstream of Kenyon cells, mushroom body output neurons show stereotypy in their responses. We found that the stereotypy is enabled by the convergence of inputs from many Kenyon cells onto an output neuron, and does not require learning. The stereotypy emerges in the total response of the Kenyon cell population using multiple odor-specific features of the projection neuron responses, benefits from the nonlinearity in the transfer function, depends on the convergence:randomness ratio, and is constrained by sparseness. Together, our results reveal the fundamental mechanisms and constraints with which convergence enables stereotypy in sensory responses despite random connectivity.

The pest species Spodoptera frugiperda, which is native to North and South America, has invaded Africa in 2016. The species consists of two strains, the corn-strain and rice-strain, which differ in their sexual communication. When we investigated populations from Benin and Nigeria, consisting of corn-strain and rice-corn-hybrid descendants, we found no strain-specific sexual communication differences. Both genotypes exhibited the same pheromone composition, consisting of around 97% (Z)-9-tetradecenyl acetate (Z9-14:Ac), 2% (Z)-7-dodecenyl acetate (Z7-12:Ac), and 1% (Z)-9-dodecenyl acetate (Z9-12:Ac), they had similar electrophysiological responses, and all mated around three hours into scotophase. However, we found geographic variation between African and American populations. The sex pheromone of African corn-strain and hybrid descendant females was similar to American rice-strain females and showed higher percentages of the male-attracting minor component Z7-12:Ac. In addition, African males exhibited the highest antennal sensitivity towards Z7-12:Ac, while American males showed highest sensitivity towards the major pheromone component Z9-14:Ac. Increasing the production of and response to the critical minor component Z7-12:Ac may reduce communication interference with other African Spodoptera species that share the same major pheromone component. The implications of our results on pheromone-based pest management strategies are discussed.

Trade-off between vision and olfaction, the fact that investment in one correlates with decreased investment in the other, has been demonstrated by a wealth of comparative studies. However, there is still no empirical evidence suggesting how these two sensory systems coevolve, i.e. simultaneously or alternatively. The "Dark-flies" (Drosophila melanogaster) constitute a unique model to investigate such relation since they have been reared in the dark since 1954, approximately 60 years (~1500 generations). To observe how vision and olfaction evolve, populations of Dark-flies were reared in normal lighting conditions for 1 (DF1G) and 65 (DF65G) generations. We measured the sizes of the visual (optic lobes, OLs) and olfactory (antennal lobes, ALs) primary centres, as well as the rest of the brain, and compared the results with the original and its genetically most similar strain (Oregon flies). We found that, whereas the ALs decreased in size, the OLs (together with the brain) increased in size in the Dark-flies returned back to the light, both in the DF1G and DF65G. These results experimentally show that trade-off between vision and olfaction occurs simultaneously, and suggests that there are possible genetic and epigenetic processes regulating the size of both optic and antennal lobes. Furthermore, although the Dark-flies were able to mate and survive in the dark with a reduced neural investment, individuals being returned to the light seem to have been selected with reinvestment in visual capabilities despite a potential higher energetic cost., Competing Interests: The authors have declared that no competing interests exist.

The general architecture of the olfactory system is highly conserved from insects to humans, but neuroanatomical and physiological differences can be observed across species. The American cockroach, inhabiting dark shelters with a rather stable olfactory landscape, is equipped with long antennae used for sampling the surrounding air-space for orientation and navigation. The antennae's exceptional length provides a wide spatial working range for odour detection; however, it is still largely unknown whether and how this is also used for mapping the structure of the olfactory environment. By selectively labelling antennal lobe projection neurons with a calcium-sensitive dye, we investigated the logic of olfactory coding in this hemimetabolous insect. We show that odour responses are stimulus specific and concentration dependent, and that structurally related odorants evoke physiologically similar responses. By using spatially confined stimuli, we show that proximal stimulations induce stronger and faster responses than distal ones. Spatially confined stimuli of the female pheromone periplanone B activate a subregion of the male macroglomerulus. Thus, we report that the combinatorial logic of odour coding deduced from holometabolous insects applies also to this hemimetabolous species. Furthermore, a fast decrease in sensitivity along the antenna, not supported by a proportionate decrease in sensillar density, suggests a neural architecture that strongly emphasizes neuronal inputs from the proximal portion of the antenna., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Females of the Queensland fruit fly (QFF), Bactrocera tryoni, are amongst the most damaging pests of horticulture in Australia and neighboring countries. Females can lay eggs into more than a hundred species of fruits and vegetables, resulting in large crop losses. Sexually mature males can be managed sustainably with traps baited with long-lasting synthetic lures, and sexually immature males and females can be attracted and killed by short-lived protein baits applied directly on surfaces, with a low success rate (< 20%). No long-lasting attractants for virgin or mated females exist. With the aim of developing a female attractant for surveillance, we collected and analyzed the odors of four ripe host fruits: orange, cherry guava, banana and feijoa. Virgin and mated female QFF were tested with gas-chromatography coupled with electro-antennographic detection to identify electrophysiologically (EAD)-active compounds. We detected 41 EAD-active compounds, with seven found common for more than one fruit. Overall, mated females responded more often and with higher intensity than virgin females. In particular, five compounds present either in cherry guava or feijoa triggered a significantly higher EAD response from mated females than from virgins. Twenty-six EAD-active compounds were selected and tested individually in a Y-tube olfactometer to measure attraction of both virgin and mated females. Behavioral responses differed significantly amongst the compounds, but not strongly between virgin and mated females. We did not find any correlation between electrophysiological and behavioral responses. Further field testing with behaviorally-active compounds is needed for the development of a new QFF female lure.

Chouioia cunea (Yang) is a pupal parasitoid wasp and this species is able to seek host insects depending on its olfactory system. However, the molecular mechanism of the olfactory system in the C. cunea is still limited. To identify putative semiochemicals bound to CcOBP2, a protein specifically expressed in antennae, 14 compounds from the pupae of H. cunea and 11 common volatile compounds from plants were selected for competitive fluorescence binding assay. The result of the binding assay showed that five compounds were able to bind toCcOBP2. The electroantennogram (EAG) demonstrated that the antennae had a significant response to the 3-Carene, a bicyclic monoterpene, and C. cunea could be obviously attracted by this compound. The behavioral response to 3- carene was dramatically weakened when CcOBP2 was specifically knocked down. The molecular docking result indicated that several amino acids especially Ile-81, Val-122, Phe-123 of CcOBP2 were responsible for binding to 3-Carene. Furthermore, there was a repellent effect on the host H. cunea with the treatment of the 3-Carene. This study illustrated that CcOBP2 might be a crucial protein involved in the olfactory signaling pathway and the 3-Carene, secreted from plants, could probably have a potential role in repelling pests as well as attracting natural enemies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bombykol (EZ) is the single component of the female sex pheromone in the silkmoth Bombyx mori. EZ alone evokes full courtship behaviors from conspecific males; however, its geometric isomer (EE) was consistently detected in the pheromone glands (PG) of 16 B. mori strains and a field population of the wild silkmoth Bombyx mandarina, which also uses EZ as the single pheromone component. We investigated the pheromonal activities of EE using a commercial hybrid strain of B. mori, Kinshu × Showa. The behavioral assay demonstrated that a 10 4 -10 5 -fold higher dose of EE than EZ was able to elicit behavioral responses from males. To elucidate whether the trace contaminant of EZ in the EE standard is responsible for these responses, we examined the responses of male antennae to EE using a gas chromatograph-electroantennographic detector system (GC-EAD). The EE, at high doses elicited marginal responses from the male antennae. We next examined antennal and behavioral responses of B. mori whose BmOR1 gene, which is responsible for the reception of bombykol, was knocked out. The knockout of BmOR1 resulted in the complete loss of antennal and behavioral responses to EE and EZ, demonstrating that if EE itself is active, it induces these responses via the incidental stimulation of BmOR1, not via the stimulation of EE-specific receptors. The existence of EE in the PG of B. mori and B. mandarina is discussed from the viewpoints of pheromone biosynthesis and the evolution of pheromone communication systems., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Mosquitoes are important vectors of disease and require sources of carbohydrates for reproduction and survival. Unlike host-related behaviors of mosquitoes, comparatively less is understood about the mechanisms involved in nectar-feeding decisions, or how this sensory information is processed in the mosquito brain. Here we show that Aedes spp. mosquitoes, including Aedes aegypti , are effective pollinators of the Platanthera obtusata orchid, and demonstrate this mutualism is mediated by the orchid's scent and the balance of excitation and inhibition in the mosquito's antennal lobe (AL). The P. obtusata orchid emits an attractive, nonanal-rich scent, whereas related Platanthera species-not visited by mosquitoes-emit scents dominated by lilac aldehyde. Calcium imaging experiments in the mosquito AL revealed that nonanal and lilac aldehyde each respectively activate the LC2 and AM2 glomerulus, and remarkably, the AM2 glomerulus is also sensitive to N,N-diethyl-meta-toluamide (DEET), a mosquito repellent. Lateral inhibition between these 2 glomeruli reflects the level of attraction to the orchid scents. Whereas the enriched nonanal scent of P. obtusata activates the LC2 and suppresses AM2, the high level of lilac aldehyde in the other orchid scents inverts this pattern of glomerular activity, and behavioral attraction is lost. These results demonstrate the ecological importance of mosquitoes beyond operating as disease vectors and open the door toward understanding the neural basis of mosquito nectar-seeking behaviors., Competing Interests: Competing interest statement: A provisional patent on the odor that mimics the orchid scent was recently filed (62/808,710)., (Copyright © 2020 the Author(s). Published by PNAS.)

There have been relatively few studies of how central synapses age in adult Drosophila melanogaster. In this study we investigate the aging of the synaptic inputs to the Giant Fiber (GF) from auditory Johnston's Organ neurons (JONs). In previously published experiments an indirect assay of this synaptic connection was used; here we describe a new, more direct assay, which allows reliable detection of the GF action potential in the neck connective, and long term recording of its responses to sound. Genetic poisoning using diphtheria toxin expressed in the GF with R68A06-GAL4 was used to confirm that this signal indeed arose from the GF and not from other descending neurons. As before, the sound-evoked action potentials (SEPs) in the antennal nerve were recorded via an electrode inserted at the base of the antenna. It was noted that an action potential in the GF elicited an antennal twitch, which in turn evoked a mechanosensory response from the JONs in the absence of sound. We then used these extracellular recording techniques in males and female of different ages to quantify the response of the JONs to a brief sound impulse, and also to measure the strength of the connection between the JONs and the GF. At no age was there any significant difference between males and females, for any of the parameters measured. The sensitivity of the JONs to a sound impulse approximately doubled between 1 d and 10 d after eclosion, which corresponds to the period when most mating is taking place. Subsequently JON sensitivity decreased with age, being approximately half as sensitive at 20 d and one-third as sensitive at 50 d, as compared to 10 d. However, the strength of the connection between the auditory input and the GF itself remained unchanged with age, although it did show some variability that could mask any small changes., Competing Interests: The authors have declared that no competing interests exist.

The cabbage stink bugs of the genus Eurydema, encompassing several oligophagous species, such as Eurydema oleracea (L.), are known to be important pests of cabbage, broccoli, and other cole crops in Europe. Despite their economic importance, the knowledge regarding the role of chemical cues in host plant selection of these species is very limited. The present investigation on E. oleracea at the adult stage revealed the use of olfaction in host plant selection of this species and demonstrated with behavioural tests that E. oleracea preferred feeding on wild Eruca sativa, rather than on Brassica oleracea. Moreover, ultrastructural data revealed the antennal sensilla of E. oleracea, encompassing single walled and double walled olfactory sensilla, and electroantennographic recordings revealed their sensitivity to several host plant VOCs from E. sativa and B. oleracea. The data shown in the present research may be useful in the development of semiochemical-based strategies or trap crops for the control of this pest in the field., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Three different pheromone binding proteins (PBPs) can typically be found in the sensilla lymph of noctuid moth antennae, but their relative contributions in perception of the sex pheromone is rarely verified in vivo. Previously, we demonstrated that SlitPBP3 plays a minor role in the sex pheromone detection in Spodoptera litura using the CRISPR/Cas9 system. In the present study, the roles of two other SlitPBPs (SlitPBP1 and SlitPBP2) are further verified using the same system. First, by co-injection of Cas9 mRNA/sgRNA into newly laid eggs, a high rate of target mutagenesis was induced, 51.5% for SlitPBP1 and 46.8% for SlitPBP2 as determined by restriction enzyme assay. Then, the homozygous SlitPBP1 and SlitPBP2 knockout lines were obtained by cross-breeding. Finally, using homozygous knockout male moths, we performed electrophysiological (EAG recording) and behavioral analyses. Results showed that knockout of either SlitPBP1 or SlitPBP2 in males decreased EAG response to each of the 3 sex pheromone components (Z9,E11-14:Ac, Z9,E12-14:Ac and Z9-14:Ac) by 53%, 60% and 63% (for SlitPBP1 knockout) and 40%, 43% and 46% (for SlitPBP2 knockout), respectively. These decreases in EAG responses were similar among 3 pheromone components, but were more pronounced in SlitPBP1 knockout males than in SlitPBP2 knockout males. Consistently, behavioral assays with the major component (Z9,E11-14:Ac) showed that SlitPBP1 knockout males responded in much lower percentages than SlitPBP2 knockout males in terms of orientation to the pheromone, along with reduction in close range behaviors such as hairpencil display and mating attempt. Taken together, this study provides direct functional evidence for the roles of SlitPBP1 and SlitPBP2, as well as their relative importance (SlitPBP1 > SlitPBP2) in the sex pheromone perception. This information is valuable in understanding mechanisms of sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The electroantennogram (EAG) is a technique used for measuring electrical signals from the antenna of an insect. Its rapid response time, quick recovery speed, and high sensitivity make it suitable for odour-tracking tasks employing mobile robots. However, its application to flying robots has not been extensively studied owing to the electrical and mechanical noises generated. In this study, we investigated the characteristics of the EAG mounted on a tethered flying quadcopter and developed a special counter-based algorithm for detecting the odour-generated responses. As the EAG response is negative, the algorithm creates a window and compares the values inside it. Once a value is smaller than the first one, the counter will increase by one and finally turns the whole signal into a clearer odour stimulated result. By experimental evaluation, the new algorithm gives a higher cross-correlation coefficient when compared with the fixed-threshold method. The result shows that the accuracy of this novel algorithm for recognising odour-evoked EAG signals from noise exceeds that of the traditional method; furthermore, the use of insect antennae as odour sensors for flying robots is demonstrated to be feasible.

The meadow moth Loxostege sticticalis is a serious agricultural pest that feeds on the leaves of many economic crops, such as sugar beet, soybean, sunflower, and potato. In addition to the rapid migration of adult moths, the collective foraging behavior of the larvae is also thought to be involved in the search for new food sources and substantially contributes to the expansion of the infested area. However, whether and how the chemical signals take part in this process remains unknown. In this study, two larva-specific expressed odorants, LstiOR5 and LstiOR6, were successfully cloned and deophanized. A heterologous study on Xenopus laevis oocytes showed that several host plant volatiles could evoke LstiOR responses in a dose-dependent manner. One herbivore-induced plant volatile (HIPV) of soybean leaves, methyl salicylate (MeSA), exerted attractive effects on the L. sticticalis larvae at all tested concentrations. Further foraging choice assays showed that the L. sticticalis larvae preferred foraged soybean leaves over unforaged leaves. When MeSA was artificially added to unforaged leaves, the unforaged leaves were preferred over the foraged leaves. In addition, GC-MS analysis demonstrated that MeSA was induced by the foraging behavior of the larvae and acted as a collective food signal in L. sticticalis. Moreover, in situ hybridization showed that LstiOR5 was highly expressed in larval antenna neurons. When LstiOR5 was silenced, both the electrophysiological response of the antenna to MeSA and the preference for foraged leaves were significantly decreased, suggesting that LstiOR5 is involved in the collective foraging behavior of L. sticticalis. Our results clarified the chemical signals that trigger the collective foraging behavior of L. sticticalis and provided more evidence for the molecular mechanism underlying the expansions of their infested areas at a peripheral olfactory sensing level. These findings could facilitate the development of potential control strategies for controlling this pest and provide a potential gene target that correlates with the collective foraging behavior of L. sticticalis, which might lead to better pest management., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Light discrimination according to colour can confer survival advantages by guiding animals towards food and shelter and away from potentially harmful situations 1,2 . Such colour-dependent behaviour can be learned or innate. Data on innate colour preference in mammals remain controversial 3 and there are limited data for simpler organisms 4-7 . Here we show that, when given a choice among blue, green and dim light, fruit flies exhibit an unexpectedly complex pattern of colour preference that changes according to the time of day. Flies show a strong preference for green in the early morning and late afternoon, a reduced green preference at midday and a robust avoidance of blue throughout the day. Genetic manipulations reveal that the peaks in green preference require rhodopsin-based visual photoreceptors and are controlled by the circadian clock. The midday reduction in green preference in favour of dim light depends on the transient receptor potential (TRP) channels dTRPA1 and Pyrexia, and is also timed by the clock. By contrast, avoidance of blue light is primarily mediated by multidendritic neurons, requires rhodopsin 7 and the TRP channel Painless, and is independent of the clock. Our findings show that several TRP channels are involved in colour-driven behaviour in Drosophila, and reveal distinct pathways of innate colour preference that coordinate the behavioural dynamics of flies in ambient light.

Reproductive investment is a central life history variable that influences all aspects of life. Hormones coordinate reproduction in multicellular organisms, but the mechanisms controlling the collective reproductive investment of social insects are largely unexplored. One important aspect of honey bee (Apis mellifera) reproductive investment consists of raising female-destined larvae into new queens by alloparental care of nurse bees in form of royal jelly provisioning. Artificial selection for commercial royal jelly production over 40 years has increased this reproductive investment by an order of magnitude. In a cross-fostering experiment, we establish that this shift in social phenotype is caused by nurse bees. We find no evidence for changes in larval signalling. Instead, the antennae of the nurse bees of the selected stock are more responsive to brood pheromones than control bees. Correspondingly, the selected royal jelly bee nurses are more attracted to brood pheromones than unselected control nurses. Comparative proteomics of the antennae from the selected and unselected stocks indicate putative molecular mechanisms, primarily changes in chemosensation and energy metabolism. We report expression differences of several candidate genes that correlate with the differences in reproductive investment. The functional relevance of these genes is supported by demonstrating that the corresponding proteins can competitively bind one previously described and one newly discovered brood pheromone. Thus, we suggest several chemosensory genes, most prominently OBP16 and CSP4, as candidate mechanisms controlling queen rearing, a key reproductive investment, in honey bees. These findings reveal novel aspects of pheromonal communication in honey bees and explain how sensory changes affect communication and lead to a drastic shift in colony-level resource allocation to sexual reproduction. Thus, pheromonal and hormonal communication may play similar roles for reproductive investment in superorganisms and multicellular organisms, respectively., (© 2019 John Wiley & Sons Ltd.)

Female Periplaneta americana cockroaches emit two cooperatively working pheromone components, periplanone-B (PB) as a long-range attractant and periplanone-A (PA) as a short-range arrestant, and males develop enlarged glomeruli for processing them separately in the first-order olfactory center. Using intracellular recordings and neuronal labelings, we found that the Turkestan cockroach, Blatta lateralis, which is phylogenetically close to P. americana but having adapted to inground habitats, has an extraordinary large glomerulus. This is caused by drastic enlargement of the PB-responsive glomerulus but not the PA-responsive glomerulus during the late nymphal stage. The output neuron from the macroglomerulus is sensitive to both PA and PB, at a dose of only 0.1 fg. Nevertheless, B. lateralis males never exhibited courtship rituals in response to the presentation of periplanones or natural sex pheromone but exhibited courtship rituals in response to antennal contact with females. Our findings indicate that the unique behavioral ecology and habitats of B. lateralis are related to the functional unification of the pheromone processing system, opposite to the functional differentiation that often underlies species diversification., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Controlling Ae. aegypti populations and the prevention of mosquito bites includes the development of monitoring, repelling and attract-and-kill strategies that are based on understanding the chemical ecology of these pests. Olfactory-mediated attraction to mammals has recently been linked to the mosquito Aedes aegypti odorant receptor Or4, which is activated by animal-released 6-Methyl-5-hepten-2-one (sulcatone). This odorant is also a major component of flower scents and may play a role outside animal-host seeking. To explore the role of this chemical cue, we looked at the interaction between sulcatone and an Or4 homolog expressed in the antennae of the strict nectar-feeding mosquito Toxorhynchites amboinensis. Using the two-electrode voltage clamp of Xenopus oocytes as a heterologous expression system, we show that this receptor is a high intensity sulcatone receptor comparable to its Aedes counterparts. We also show that OR4 is activated by other aliphatic ketones and is inhibited by DEET. This pharmacological characterization suggests that sulcatone may be operating in more than one context in the Culicidae family., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The morphology and ultrastructure of the olfactory sensilla on the antennae and maxillary palps were investigated through scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and their responses to five volatile compounds were measured using electroantenogram (EAG) and electropalpogram (EPG) techniques in the pumpkin fruit fly, Bactrocera depressa (Shiraki; Diptera: Tephritidae). Male and female B. depressa displayed distinct morphological types of olfactory sensilla in the antennae and maxillary palps, with predominant populations of trichoid, basiconic, and coeloconic sensilla. Basiconic sensilla, the most abundant type of olfactory sensilla in the antennae, could be further classified into two different types. In contrast, the maxillary palps exhibited predominant populations of a single type of curved basiconic sensilla. High-resolution SEM observation revealed the presence of multiple nanoscale wall-pores on the cuticular surface of trichoid and basiconic sensilla, indicating that their primary function is olfactory. In contrast, coeloconic sensilla displayed several longitudinal grooves around the sensillum peg. The TEM observation of individual antennal olfactory sensilla indicates that the basiconic sensilla are thin-walled, while the trichoid sensilla are thick-walled. The profile of EAG responses of male B. depressa was different from their EPG response profile, indicating that the olfactory function of maxillary palps is different from that of antennae in this species. The structural and functional variation in the olfactory sensilla between antennae and maxillary palps suggests that each plays an independent role in the perception of olfactory signals in B. depressa., (© 2019 Wiley Periodicals, Inc.)