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.

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.

Microscopy and biomimetics: the NanoSuit® method and image retrieval platform.

This review aims to clarify a suitable method towards achieving next-generation sustainability. As represented by the term 'Anthropocene', the Earth, including humans, is entering a critical era; therefore, science has a great responsibility to solve it. Biomimetics, the emulation of the models, systems and elements of nature, especially biological science, is a powerful tool to approach sustainability problems. Microscopy has made great progress with the technology of observing biological and artificial materials and its techniques have been continuously improved, most recently through the NanoSuit® method. As one of the most important tools across many facets of research and development, microscopy has produced a large amount of accumulated digital data. However, it is difficult to extract useful data for making things as biomimetic ideas despite a large amount of biological data. Here, we would like to find a way to organically connect the indispensable microscopic data with the new biomimetics to solve complex human problems.

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.

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.

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.

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.

Functional characterization of olfactory receptors in the Oriental fruit fly Bactrocera dorsalis that respond to plant volatiles.

The Oriental fruit fly, Bactrocera dorsalis, is a highly destructive pest of various fruits. The reproductive and host-finding behaviors of this species are affected by several plant semiochemicals that are perceived through chemosensory receptors. However, the chemosensory mechanisms by which this perception occurs have not been fully elucidated. We conducted RNA sequencing analysis of the chemosensory organs of B. dorsalis to identify the genes coding for chemosensory receptors. We identified 60 olfactory receptors (ORs), 17 gustatory receptors and 23 ionotropic receptors-including their homologs and variants-from the transcriptome of male antennae and proboscises. We functionally analyzed ten ORs co-expressed with the obligatory co-receptor ORCO in Xenopus oocytes to identify their ligands. We tested 24 compounds including attractants for several Bactrocera species and volatiles from the host fruits of B. dorsalis. We found that BdorOR13a co-expressed with ORCO responded robustly to 1-octen-3-ol. BdorOR82a co-expressed with ORCO responded significantly to geranyl acetate, but responded weakly to farnesenes (a mixture of isomers) and linalyl acetate. These four compounds were subsequently subjected to behavioral bioassays. When each of the aforementioned compound was presented in combination with a sphere model as a visual cue to adult flies, 1-octen-3-ol, geranyl acetate, and farnesenes significantly enhanced landing behavior in mated females, but not in unmated females or males. These results suggest that the ORs characterized in the present study are involved in the perception of plant volatiles that affect host-finding behavior in B. dorsalis.

Increasing cell-device adherence using cultured insect cells for receptor-based biosensors.

Field-effect transistor (FET)-based biosensors have a wide range of applications, and a bio-FET odorant sensor, based on insect (Sf21) cells expressing insect odorant receptors (ORs) with sensitivity and selectivity, has emerged. To fully realize the practical application of bio-FET odorant sensors, knowledge of the cell-device interface for efficient signal transfer, and a reliable and low-cost measurement system using the commercial complementary metal-oxide semiconductor (CMOS) foundry process, will be indispensable. However, the interfaces between Sf21 cells and sensor devices are largely unknown, and electrode materials used in the commercial CMOS foundry process are generally limited to aluminium, which is reportedly toxic to cells. In this study, we investigated Sf21 cell-device interfaces by developing cross-sectional specimens. Calcium imaging of Sf21 cells expressing insect ORs was used to verify the functions of Sf21 cells as odorant sensor elements on the electrode materials. We found that the cell-device interface was approximately 10 nm wide on average, suggesting that the adhesion mechanism of Sf21 cells may differ from that of other cells. These results will help to construct accurate signal detection from expressed insect ORs using FETs.

Cell-Based Odorant Sensor Array for Odor Discrimination Based on Insect Odorant Receptors.

The olfactory system of living organisms can accurately discriminate numerous odors by recognizing the pattern of activation of several odorant receptors (ORs). Thus, development of an odorant sensor array based on multiple ORs presents the possibility of mimicking biological odor discrimination mechanisms. Recently, we developed novel odorant sensor elements with high sensitivity and selectivity based on insect OR-expressing Sf21 cells that respond to target odorants by displaying increased fluorescence intensity. Here we introduce the development of an odorant sensor array composed of several Sf21 cell lines expressing different ORs. In this study, an array pattern of four cell lines expressing Or13a, Or56a, BmOR1, and BmOR3 was successfully created using a patterned polydimethylsiloxane film template and cell-immobilizing reagents, termed biocompatible anchor for membrane (BAM). We demonstrated that BAM could create a clear pattern of Sf21 sensor cells without impacting their odorant-sensing performance. Our sensor array showed odorant-specific response patterns toward both odorant mixtures and single odorant stimuli, allowing us to visualize the presence of 1-octen-3-ol, geosmin, bombykol, and bombykal as an increased fluorescence intensity in the region of Or13a, Or56a, BmOR1, and BmOR3 cell lines, respectively. Therefore, we successfully developed a new methodology for creating a cell-based odorant sensor array that enables us to discriminate multiple target odorants. Our method might be expanded into the development of an odorant sensor capable of detecting a large range of environmental odorants that might become a promising tool used in various applications including the study of insect semiochemicals and food contamination.

Identification of repellent odorants to the body louse, Pediculus humanus corporis, in clove essential oil.

The control of body lice is an important issue for human health and welfare because lice act as vectors of disease such as typhus, relapsing fever, and trench fever. Body lice exhibit avoidance behavior to some essential oils, including clove essential oil. Therefore, odorants containing clove essential oil components may potentially be useful in the development of repellents to body lice. However, such odorants that induce avoidance behavior in body lice have not yet been identified from clove essential oil. Here, we established an analysis method to evaluate the avoidance behavior of body lice to specific odorants. The behavioral analysis of the body lice in response to clove essential oil and its constituents revealed that eugenol, a major component of clove essential oil, has strong repellent effect on body lice, whereas the other components failed to induce obvious avoidance behavior. A comparison of the repellent effects of eugenol with those of other structurally related odorants revealed possible moieties that are important for the avoidance effects to body lice. The repellent effect of eugenol to body lice was enhanced by combining it with the other major component of clove essential oil, ß-caryophyllene. We conclude that a synthetic blend of eugenol and ß-caryophyllene is the most effective repellent to body lice. This finding will be valuable as the potential use of eugenol as body lice repellent.

Targeted disruption of a single sex pheromone receptor gene completely abolishes in vivo pheromone response in the silkmoth.

Male moths use species-specific sex pheromones to identify and orientate toward conspecific females. Odorant receptors (ORs) for sex pheromone substances have been identified as sex pheromone receptors in various moth species. However, direct in vivo evidence linking the functional role of these ORs with behavioural responses is lacking. In the silkmoth, Bombyx mori, female moths emit two sex pheromone components, bombykol and bombykal, but only bombykol elicits sexual behaviour in male moths. A sex pheromone receptor BmOR1 is specifically tuned to bombykol and is expressed in specialized olfactory receptor neurons (ORNs) in the pheromone sensitive long sensilla trichodea of male silkmoth antennae. Here, we show that disruption of the BmOR1 gene, mediated by transcription activator-like effector nucleases (TALENs), completely removes ORN sensitivity to bombykol and corresponding pheromone-source searching behaviour in male moths. Furthermore, transgenic rescue of BmOR1 restored normal behavioural responses to bombykol. Our results demonstrate that BmOR1 is required for the physiological and behavioural response to bombykol, demonstrating that it is the receptor that mediates sex pheromone responses in male silkmoths. This study provides the first direct evidence that a member of the sex pheromone receptor family in moth species mediates conspecific sex pheromone information for sexual behaviour.

Novel cell-based odorant sensor elements based on insect odorant receptors.

Development of cell-based odorant sensor elements combined not only high degree of sensitivity and selectivity but also long-term stability is crucial for their practical applications. Here we report the development of a novel cell-based odorant sensor element that sensitively and selectively detects odorants and displays increased fluorescent intensities over a long period of time. Our odorant sensor elements, based on Sf21 cell lines expressing insect odorant receptors, are sensitive to the level of several tens of parts per billion in solution, can selectively distinguish between different types of odorants based on the odorant selectivity intrinsic to the expressed receptors, and have response times of approximately 13s. Specifically, with the use of Sf21 cells and insect odorant receptors, we demonstrated that the established cell lines stably expressing insect odorant receptors are able to detect odorants with consistent responsiveness for at least 2 months, thus exceeding the short life-span normally associated with cell-based sensors. We also demonstrated the development of a compact odorant sensor chip by integrating the established insect cell lines into a microfluidic chip. The methodology we established in this study, in conjunction with the large repertoire of insect odorant receptors, will aid in the development of practical cell-based odorant sensors for various applications, including food administration and health management.

Pheromone responsiveness threshold depends on temporal integration by antennal lobe projection neurons.

The olfactory system of male moths has an extreme sensitivity with the capability to detect and recognize conspecific pheromones dispersed and greatly diluted in the air. Just 170 molecules of the silkmoth (Bombyx mori) sex pheromone bombykol are sufficient to induce sexual behavior in the male. However, it is still unclear how the sensitivity of olfactory receptor neurons (ORNs) is relayed through the brain to generate high behavioral responsiveness. Here, we show that ORN activity that is subthreshold in terms of behavior can be amplified to suprathreshold levels by temporal integration in antennal lobe projection neurons (PNs) if occurring within a specific time window. To control ORN inputs with high temporal resolution, channelrhodopsin-2 was genetically introduced into bombykol-responsive ORNs. Temporal integration in PNs was only observed for weak inputs, but not for strong inputs. Pharmacological dissection revealed that GABAergic mechanisms inhibit temporal integration of strong inputs, showing that GABA signaling regulates PN responses in a stimulus-dependent fashion. Our results show that boosting of the PNs' responses by temporal integration of olfactory information occurs specifically near the behavioral threshold, effectively defining the lower bound for behavioral responsiveness.