Active Tracking of Temporally Changing Gas-Phase Odor Mixture Using an Array of Cells Expressing Olfactory Receptors.

A cell expressing an olfactory receptor (OR) exhibits excellent odorant detection ability and thus is widely applied in odor biosensors. Most of those biosensors, however, could detect only liquid-phase nonchanging single-component odorants. In this paper, we raised up an odor biosensor for the active tracking of temporally changing gas-phase odor mixture by an array of cells expressing ORs. A thin stable liquid film covered the cell, thus allowing gas-phase odorants to penetrate. The online image processing generated individual cell brightness data which were used to compute the biosensor response. Based on the obtained responses, we adjusted the known odor components to be similar with the unknown odor. The function of our biosensor was validated by tracking the variable single-component odorant or the binary odor mixture. The influence from the sensor drift could be overcome by comparing the adjacent unknown and known odor responses. In the odor mixture quantification, adding the OR label to mixed cells and then quantifying separately (named as the pre-label method) was more efficient, while directly using the cell response pattern (named as the label-free method) was still capable even if the OR odor had cross-sensitivity.

Xenopus laevis Oocyte Array Fluidic Device Integrated with Microelectrodes for A Compact Two-Electrode Voltage Clamping System.

We report on a compact two-electrode voltage clamping system composed of microfabricated electrodes and a fluidic device for Xenopus laevis oocytes. The device was fabricated by assembling Si-based electrode chips and acrylic frames to form fluidic channels. After the installation of Xenopus oocytes into the fluidic channels, the device can be separated in order to measure changes in oocyte plasma membrane potential in each channel using an external amplifier. Using fluid simulations and experiments, we investigated the success rates of Xenopus oocyte arrays and electrode insertion with respect to the flow rate. We successfully located each oocyte in the array and detected oocyte responses to chemical stimuli using our device.

Spike frequency adaptation facilitates the encoding of input gradient in insect olfactory projection neurons.

The olfactory system in insects has evolved to process the dynamic changes in the concentration of food odors or sex pheromones to localize the nutrients or conspecific mating partners. Experimental studies have suggested that projection neurons (PNs) in insects encode not only the stimulus intensity but also its rate-of-change (input gradient). In this study, we aim to develop a simple computational model for a PN to understand the mechanism underlying the coding of the rate-of-change information. We show that the spike frequency adaptation is a potential key mechanism for reproducing the phasic response pattern of the PN in Drosophila. We also demonstrate that this adaptation mechanism enables the PN to encode the rate-of-change of the input firing rate. Finally, our model predicts that the PN exhibits the intensity-invariant response for the pulse and ramp odor stimulus. These results suggest that the developed model is useful for investigating the coding principle underlying olfactory information processing in insects.

Use of Visual Information by Ant Species Occurring in Similar Urban Anthropogenic Environments.

Many insects, including ants, are known to respond visually to conspicuous objects. In this study, we compared orientation in an arena containing only a black target beacon as local information in six species of ants of widely varying degree of phylogenic relatedness, foraging strategy, and eye morphology (Aphaenogaster, Brachyponera, Camponotus, Formica, and two Lasius spp.), often found associated in similar urban anthropogenic habitats. Four species of ants displayed orientation toward the beacon, with two orienting toward it directly, while the other two approached it via convoluted paths. The two remaining species did not show any orientation with respect to the beacon. The results did not correlate with morphological parameters of the visual systems and could not be fully interpreted in terms of the species' ecology, although convoluted paths are linked to higher significance of chemical signals. Beacon aiming was shown to be an innate behavior in visually naive Formica workers, which, however, were less strongly attracted to the beacon than older foragers. Thus, despite sharing the same habitats and supposedly having similar neural circuits, even a very simple stimulus-related behavior in the absence of other information can differ widely in ants but is likely an ancestral trait retained especially in species with smaller eyes. The comparative analysis of nervous systems opens the possibility of determining general features of circuits responsible for innate and possibly learned attraction toward particular stimuli.

Low Surface Potential with Glycoconjugates Determines Insect Cell Adhesion at Room Temperature.

Cell-coupled field-effect transistor (FET) biosensors have attracted considerable attention because of their high sensitivity to biomolecules. The use of insect cells (Sf21) as a core sensor element is advantageous due to their stable adhesion to sensors at room temperature. Although visualization of the insect cell-substrate interface leads to logical amplification of signals, the spatiotemporal processes at the interfaces have not yet been elucidated. We quantitatively monitored the adhesion dynamics of Sf21 using interference reflection microscopy (IRM). Specific adhesion signatures with ring-like patches along the cellular periphery were detected. A combination of zeta potential measurements and lectin staining identified specific glycoconjugates with low electrostatic potentials. The ring-like structures were disrupted after cholesterol depletion, suggesting a raft domain along the cell periphery. Our results indicate dynamic and asymmetric cell adhesion is due to low electrostatic repulsion with fluidic sugar rafts. We envision the logical design of cell-sensor interfaces with an electrical model that accounts for actual adhesion interfaces.

Modeling the musculoskeletal system of an insect thorax for flapping flight.

Indirect actuation of the wings via thoracic deformation is a unique mechanism widely observed in flying insect species. The physical properties of the thorax have been intensively studied in terms of their ability to efficiently generate wingbeats. The basic mechanism of indirect wing actuation is generally explained as a lever model on a cross-sectional plane, where the dorsoventral movement of the mesonotum (dorsal exoskeleton of the mesothorax) generated by contractions of indirect muscles actuates the wing. However, the model considers the mesonotum as an ideal flat plane, whereas the mesonotum is hemispherical and becomes locally deformed during flight. Furthermore, the conventional model is two-dimensional; therefore, three-dimensional wing kinematics by indirect muscles have not been studied to date. In this study, we develop structural models of the mesonotum and mesothorax of the hawkmothAgrius convolvuli, reconstructed from serial cross-sectional images. External forces are applied to the models to mimic muscle contraction, and mesonotum deformation and wing trajectories are analyzed using finite element analysis. We find that applying longitudinal strain to the mesonotum to mimic strain by depressor muscle contraction reproduces local deformation comparable to that of the thorax during flight. Furthermore, the phase difference of the forces applied to the depressor and elevator muscles changes the wing trajectory from a figure eight to a circle, which is qualitatively consistent with the tethered flight experiment. These results indicate that the local deformation of the mesonotum due to its morphology and the thoracic deformation via indirect power muscles can modulate three-dimensional wing trajectories.

Extending lifetime of gas-phase odor biosensor using liquid thickness control and liquid exchange.

In recent few years, researchers utilized cell expressing olfactory receptor for vapor detection under various sensing mechanisms. Those olfactory systems, however, have relatively short lifetime due to the dry out of aqueous solution covering the cell. In this paper, we came up with a feedback control structure composed of an impedance measurement circuit, a microcontroller and two syringe pumps for maintaining thin liquid layer above cell. Cell lifetime was improved from less than 40 min to longer than 75 min when liquid film control was introduced. However, the biosensor lifetime remained similar between with or without liquid thickness control. Then, we added liquid exchange to further extend the lifetime of our odor biosensor. Minimal liquid exchange speed was able to significantly extend the biosensor lifetime. Meanwhile, faster liquid exchange speed resulted in better sensor responses. Furthermore, the enhancement acquired from intermittent liquid exchange was compared with continuous one. In this study, the lifetime of odor biosensor was extended to more than 3 h whereas it was less than half an hour without liquid thickness control. We believe the methodology we established in this paper will facilitate gas phase odor biosensor in continuous monitoring of target substances.

Pheromone binding protein is involved in temporal olfactory resolution in the silkmoth.

Male moths utilize spatio-temporal female sex pheromone information to orient toward conspecific females. Pheromones are distributed as discontinuous plumes owing to air turbulence; thus, efficient tracking of intermittent stimuli is expected to require a high temporal resolution. Here, using pheromone binding protein (BmPBP1)-knockout silkmoths, we showed that a loss of functional PBP lowered the temporal sensory resolution of male antennae. This altered temporal resolution resulted in significantly reduced straight walking and longer turning behavior, which respectively occurred when males detected and lost contact with pheromones, indicating that temporal resolution was also lowered at the behavioral level. BmPBP1-knockout males required significantly longer time than wild-type males in locating pheromone sources and female moths. Our results suggest that BmPBP1 plays a critical role in determining olfactory response kinetics. Accordingly, high temporal olfactory and behavioral resolutions, as shaped by PBP, are essential for tracking pheromone plumes and locating females efficiently.

Electroantennography-based Bio-hybrid Odor-detecting Drone using Silkmoth Antennae for Odor Source Localization.

Small drones with chemical or biosensor devices that can detect airborne odorant molecules have attracted considerable attention owing to their applicability in environmental and security monitoring and search-and-rescue operations. Small drones with commercial metal-oxide-semiconductor (MOX) gas sensors have been developed for odor source localization; however, their real-time-odor-detection performance has proven inadequate. However, biosensing technologies based on insect olfactory systems exhibit relatively high sensitivity, selectivity, and real-time response with respect to odorant molecules compared to commercial MOX gas sensors. In such devices, excised insect antennae function as portable odorant biosensor elements and have been found to deliver excellent sensing performance. This study presents experimental protocols for odorant-molecule detection in the air using a small autonomous bio-hybrid drone based on a mountable electroantennography (EAG) device incorporating silkmoth antennae. We developed a mountable EAG device including sensing/processing parts with a Wi-Fi module. The device was equipped with a simple sensor enclosure to enhance the sensor directivity. Thus, odor source localization was conducted using the spiral-surge algorithm, which does not assume an upwind direction. The experimental bio-hybrid odor-detecting drone identified real-time odorant-concentration differences in a pseudo-open environment (outside a wind tunnel) and localized the source. The developed drone and associated system can serve as an efficient odorant molecule-detection tool and a suitable flight platform for developing odor source localization algorithms owing to its high programmability.

Identification of Exploration and Exploitation Balance in the Silkmoth Olfactory Search Behavior by Information-Theoretic Modeling.

Insects search for and find odor sources as their basic behaviors, such as when looking for food or a mate. This has motivated research to describe how they achieve such behavior under turbulent odor plumes with a small number of neurons. Among different insects, the silk moth has been studied owing to its clear motor response to olfactory input. In past studies, the "programmed behavior" of the silk moth has been modeled as the average duration of a sequence of maneuvers based on the duration of periods without odor hits. However, this model does not fully represent the fine variations in their behavior. In this study, we used silk moth olfactory search trajectories from an experimental virtual reality device. We achieved an accurate input by using optogenetic silk moths that react to blue light. We then modeled such trajectories as a probabilistic learning agent with a belief of possible source locations. We found that maneuvers mismatching the programmed behavior are related to larger entropy decrease, that is, they are more likely to increase the certainty of the belief. This implies that silkmoths include some stochasticity in their search policy to balance the exploration and exploitation of olfactory information by matching or mismatching the programmed behavior model. We believe that this information-theoretic representation of insect behavior is important for the future implementation of olfactory searches in artificial agents such as robots.

Binary mixture quantification using cell-based odor biosensor system with active sensing.

Organisms perceive odorants in the environment through the use of a large number of olfactory receptors. Various odor biosensors have been researched and developed in order to mimic this olfactory mechanism. This study examines the quantification of odorant concentrations through the use of a sensor array comprised of several types of cell-based odor sensors expressing insect olfactory receptors with nonlinear characteristics. The sensor system utilized an active sensing method in order to compare the responses of a target odorant and a prepared odorant in determining the relative concentration of the target odorant. By combining an active sensing method with a real-time reference method in which the target odorant was measured every time the prepared odorant was measured, the relative concentrations were successfully determined even when the response fluctuation was large or odorant sensor cell responses varied as measurement time increased. For proof of concept purposes, the study primarily focused on quantifying odorant concentrations composed of one or two odorant components. It was confirmed that an algorithm to find the optimal relative odorant concentration among a limited number of odorant concentrations is achievable. Though this study is still in the initial stage of the developing odor sensors and has many challenges, it can provide insight into paving the way towards a new type of odor biosensor with active sensing.

Highly effective volatile organic compound dissolving strategy based on mist atomization for odorant biosensors.

The detection of volatile organic compound (VOC) mixtures is crucial in the medical and security fields. Receptor-based odorant biosensors sensitively and selectively detect odorant molecules in a solution; however, odorant molecules generally exist as VOCs in the air and exhibit poor water solubility. Therefore, techniques that enable the dissolution of poorly water-soluble VOCs using portable systems are essential for practical biosensors' applications. We previously proposed a VOC dissolution method based on water atomization to increase the surface area via the generation of fine bubbles, as a proof-of-concept; however, the system was lab-based (non-mobile) and the dissolution was limited to one VOC. In this study, we established a highly effective VOC dissolution method based on mist atomization that can be used in the field. This new method demonstrated a rapid dissolution potential of a sparsely-soluble VOC mixture with various functional groups in distilled water (DW) within 1 min, without the use of any organic solvents. Calcium imaging revealed that odorant receptor 13a-expressing Sf21 cells (Or13a cells) responded to 1-octen-3-ol in the mixture. Further, we successfully developed a field-deployable prototype vacuum and dissolution system with a simple configuration that efficiently captured and rapidly dissolved airborne 1-octen-3-ol in DW. This study proposes a field-deployable system that is appropriate for solubilizing various airborne odorant molecules and therefore is a practical strategy to use in the context of odorant biosensors.

3D-Printed Bubble-Free Perfusion Cartridge System for Live-Cell Imaging.

The advent of 3D-printing technologies has had a significant effect on the development of medical and biological devices. Perfusion chambers are widely used for live-cell imaging in cell biology research; however, air-bubble invasion is a pervasive problem in perfusion systems. Although 3D printing allows the rapid fabrication of millifluidic and microfluidic devices with high resolution, little has been reported on 3D-printed fluidic devices with bubble trapping systems. Herein, we present a 3D-printed millifluidic cartridge system with bent and flat tapered flow channels for preventing air-bubble invasion, irrespective of bubble volume and without the need for additional bubble-removing devices. This system realizes bubble-free perfusion with a user-friendly interface and no-time-penalty manufacturing processes. We demonstrated the bubble removal capability of the cartridge by continually introducing air bubbles with different volumes during the calcium imaging of Sf21 cells expressing insect odorant receptors. Calcium imaging was conducted using a low-magnification objective lens to show the versatility of the cartridge for wide-area observation. We verified that the cartridge could be used as a chemical reaction chamber by conducting protein staining experiments. Our cartridge system is advantageous for a wide range of cell-based bioassays and bioanalytical studies, and can be easily integrated into portable biosensors.

Insect-machine hybrid robot.

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.

Pheromonal activities of the bombykol isomer, (10E,12E)-10,12-hexadecadien-1-ol, in the pheromone gland of the silkmoth Bombyx mori.

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 104-105-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.

Functional characterization of olfactory receptors in three Dacini fruit flies (Diptera: Tephritidae) that respond to 1-nonanol analogs as components in the rectal glands.

Dacini fruit flies (Tephritidae: Diptera), including destructive pest species, are strongly affected in their reproductive behaviors by semiochemicals. Notably, male lures have been developed for pest management e.g., aromatic compounds for the Oriental fruit fly Bactrocera dorsalis and the melon fruit fly Zeugodacus cucurbitae; terpenic α-ionone analogs for the solanaceous fruit fly, B. latifrons. Other than those specific male attractants, 1-nonanol analogs have been noticed as major aliphatic components in the male rectal gland, which is considered as a secretory organ of male sex pheromones. Although multiple semiochemicals associated with the life cycle of Dacini fruit flies have been identified, their behavioral role(s) and chemosensory mechanisms by which the perception occurs have not been fully elucidated. In this study, we conducted RNA sequencing analysis of the chemosensory organs of B. latifrons and Z. cucurbitae to identify the genes coding for chemosensory receptors. Because the skeletons of male attractants are different among Dacini fruit fly species, we analyzed phylogenetic relationships of candidate olfactory receptors (ORs) among the three species. We found that the OR phylogeny reflects the taxonomic relationships of the three species. We further characterized functional properties of OR74a in the three Dacini species to the 1-nonanol analogs related to components in the rectal glands. The three OR74a homologs responded to 1-nonanol, but their sensitivities differed from each other. The OR74a homologs identified from B. dorsalis and Z. cucurbitae responded significantly to 6-oxo-1-nonanol, but not to 1,3-nonanediol and nonyl acetate, indicating similar binding properties of the homologous ORs.

Morphology and physiology of olfactory neurons in the lateral protocerebrum of the silkmoth Bombyx mori.

Insect olfaction is a suitable model to investigate sensory processing in the brain. Olfactory information is first processed in the antennal lobe and is then conveyed to two second-order centres-the mushroom body calyx and the lateral protocerebrum. Projection neurons processing sex pheromones and plant odours supply the delta area of the inferior lateral protocerebrum (∆ILPC) and lateral horn (LH), respectively. Here, we investigated the neurons arising from these regions in the brain of the silkmoth, Bombyx mori, using mass staining and intracellular recording with a sharp glass microelectrode. The output neurons from the ∆ILPC projected to the superior medial protocerebrum, whereas those from the LH projected to the superior lateral protocerebrum. The dendritic innervations of output neurons from the ∆ILPC formed a subdivision in the ∆ILPC. We discuss pathways for odour processing in higher order centres.

Dropping Counter: A Detection Algorithm for Identifying Odour-Evoked Responses from Noisy Electroantennograms Measured by a Flying Robot.

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.

Analysis of the role of wind information for efficient chemical plume tracing based on optogenetic silkworm moth behavior.

Many animals use olfactory information to search for feeding areas and other individuals in real time and with high efficiency. We focus on the chemical plume tracing (CPT) ability of male silkworm moths and investigate an efficient CPT strategy for an autonomous robot. In the case of flying insects, the wind direction is an important factor in CPT, because the wind carries odors amongst other environmental information. However, whether the same phenomenon occurs in the walking silkworm moth has not been investigated. Therefore, we examine how the silkworm moth uses wind information during CPT. To accurately investigate the response to the wind direction, we introduce an optogenetic approach that replaces the odor stimulation with light stimulation, allowing us to separate the 'wind stimulus' from the 'odor stimulus'. We examine how the moth uses wind direction information in a biological experiment, and find that the movement speed is significantly reduced when the wind speed is relatively fast (1.0 m s-1). By implementing this phenomenon in an autonomous robot, we can improve the successful search rate over that of the conventional moth-inspired algorithm. Regarding the search time, the proposed algorithm finds the odor source faster in a low-frequency odorant emission environment, whereas the search is slower than the conventional method when the odor frequency is higher. Therefore, switching from the use of wind direction information to odor information according to the frequency with which the odor is encountered leads to efficient CPT performance.

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

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