Male Silkmoth Research Articles

Evidence for a role of SNMP2 and antennal support cells in sensillum lymph clearance processes of moth pheromone-responsive sensilla

In insect antenna, following the activation of olfactory sensory neurons, odorant molecules are inactivated by enzymes in the sensillum lymph. How the inactivation products are cleared from the sensillum lymph is presently unknown. Here we studied the role of support cells (SCs) and the so-called sensory neuron membrane protein 2 (SNMP2), a member of the CD36 family of lipid transporters abundantly expressed in SCs, in sensillum lymph clearance processes in the moths Heliothis virescens and Bombyx mori. In these species, the sex pheromone components are inactivated to long-chain fatty acids. To approach a role of SNMP2 in the removal of such inactivation products, we analyzed the uptake of a fluorescent long-chain fatty acid analog into a newly generated HvirSNMP2-expressing cell line. We found an increased uptake of the analog into SNMP2-cells compared to control cells, which could be blocked by the CD36 protein inhibitor, SSO. Furthermore, analyses of sensilla from antenna treated with the fatty acid analog indicated that SNMP2-expressing SCs are able to take up fatty acids from the sensillum lymph. In addition, sensilla from SSO-pretreated antenna of B. mori showed reduced removal of the fluorescent analog from the sensillum lymph. Finally, we revealed that SSO pretreatment of male silkmoth antenna significantly prolonged the duration of the female pheromone-induced wing-fluttering behavior, possibly as a result of impaired lymph clearance processes. Together our findings in H. virescens and B. mori support a pivotal role of olfactory SCs in sensillum lymph maintenance processes and suggest an integral role of SNMP2 in the removal of lipophilic “waste products” such as fatty acids resulting from sex pheromone inactivation.

Analysis of Odor-Tracking Performance of Silk Moth Using a Sensory-Motor Intervention System.

Animals can adaptively behave in different environmental conditions by converting environmental information obtained from their sensory organs into actions. This sensory-motor integration enables the accomplishment of various tasks and is essential for animal survival. This sensory-motor integration also plays an important role in localization to females, relying on sex pheromones floating in space. In this study, we focused on the localization behavior of the adult male silk moth, Bombyx mori. We investigated sensory-motor integration against time delay using odor plume tracking performance as an index when we set a certain time delay for the sensory and motor responses. Given that it is difficult to directly intervene in the sensory and motor functions of the silk moth, we constructed an intervention system based on a mobile behavior measurement system controlled by them. Using this intervention system, not only can timing the detection of the odor in the environment and timing the presentation of the odor to the silk moth be manipulated, but timing the reflection of the movement of the silk moth can also be manipulated. We analyzed the extent to which the localization strategy of the silk moth could tolerate sensory delays by setting a delay to the odor presentation. We also evaluated behavioral compensation by odor sensory feedback by setting a delay to the motor. The results of the localization experiment have shown that the localization success rate did not decrease when there was a motor delay. However, when there was a sensory delay, the success rate decreased depending on the time delay. Analysis of the change in behavior after detection of the odor stimulus has shown that the movement was more linear when we set a motor delay. However, the movement was accompanied by a large rotational movement when there was a delay in the sensory input. This result has suggested that behavior is compensated for the delay in motor function by feedback control of odor sensation, but not when accompanied by sensory delay. To compensate for this, the silk moth may acquire appropriate information from the environment by making large body movements.

Juvenile hormone suppresses the FoxO-takeout axis to shorten longevity in male silkworm

Juvenile hormone (JH) plays a crucial endocrine regulatory role in insect metamorphosis, reproduction, and longevity in multiple organisms, such as flies, honeybees, and migratory monarch butterflies. However, the molecular mechanism of JH affecting longevity remains largely unknown. In this study, we showed that JH III and its analog methoprene shortened the survival days significantly in the adulthood of male silkworm. At the same time, the allatostatin, a neuropeptide that inhibits the secretion of JH by the corpora allata, could extend the survival days dramatically after adult eclosion in male silkmoth. Interestingly, a central pro-longevity FoxO transcription factor was reduced upon JH stimulation in silkworm individuals and BmN-SWU1 cells. Furthermore, the analysis of the upstream sequence of the FoxO gene identified a JH response element which suggested that FoxO might be regulated as a target of JH. Surprisingly, we identified a Bmtakeout (BmTO) gene that encodes a JH-binding protein and contains a FoxO response element. As expected, FoxO overexpression and knockdown up- and down-regulated the expression of BmTO respectively, indicating that BmTO functions as a FoxO target. BmTO overexpression could release the inhibitory effect of JH on the BmFoxO gene by reducing JH bioavailability to block its signal transduction. Collectively, these results may provide insights into the mechanism of the JH-FoxO-TO axis in aging research and pest control.

Robust Moth-Inspired Algorithm for Odor Source Localization Using Multimodal Information.

Odor-source localization, by which one finds the source of an odor by detecting the odor itself, is an important ability to possess in order to search for leaking gases, explosives, and disaster survivors. Although many animals possess this ability, research on implementing olfaction in robotics is still developing. We developed a novel algorithm that enables a robot to localize an odor source indoors and outdoors by taking inspiration from the adult male silk moth, which we used as the target organism. We measured the female-localization behavior of the silk moth by using a virtual reality (VR) system to obtain the relationship between multiple sensory stimuli and behavior during the localization behavior. The results showed that there were two types of search active and inactive depending on the direction of odor and wind detection. In an active search, the silk moth moved faster as the odor-detection frequency increased, whereas in the inactive search, they always moved slower under all odor-detection frequencies. This phenomenon was constructed as a robust moth-inspired (RMI) algorithm and implemented on a ground-running robot. Experiments on odor-source localization in three environments with different degrees of environmental complexity showed that the RMI algorithm has the best localization performance among conventional moth-inspired algorithms. Analysis of the trajectories showed that the robot could move smoothly through the odor plume even when the environment became more complex. This indicates that switching and modulating behavior based on the direction of odor and wind detection contributes to the adaptability and robustness of odor-source localization.

Male silk moths for the post-mating activities of female Silkmoth Bombyx mori L.

Male silk moths for the post-mating activities of female Silkmoth Bombyx mori L.

Learning a Generic Olfactory Search Strategy From Silk Moths by Deep Inverse Reinforcement Learning

Despite their simple nervous systems, insects efficiently search for and find sources of odorants. Hence, it is necessary to model and implement such behavior in artificial agents (robots), to enable them to detect dangerous substances such as drugs, gas leaks, and explosives. Previous studies have approached behavioral modeling with either statistical or machine-learning methods. In this study, we determined the behavior trajectories of male silk moths using a virtual reality (VR) system. We then modeled these trajectories as a Markov decision process (MDP) and employed inverse reinforcement learning (IRL) to learn their reward function. Furthermore, we estimated the optimal policy from the learned reward function. We then conducted olfactory search simulations and determined that the IRL-based policy could locate odor sources with a high success rate. This was also investigated under environmental conditions different from those faced by real moths on the VR system. The obtained results indicate that IRL can generically represent olfactory search strategies that are adaptable to various environments.

Real-Time Odor Discrimination Using Single Antenna of Insect

In this letter, a system that discriminates between two odorants in real-time with a single antenna of an insect was constructed. In previous studies, odorants were discriminated through arraying multiple types of sensors. However, the use of a sensor array enlarges the entire system and requires complex signal processing, which makes it difficult to mount the system on a quadcopter with limited payloads or an autonomous robot with low computational power. On the other hand, the antenna of an insect is composed of several olfactory receptors; thus, a single antenna responds to multiple odorants. Therefore, a system for discriminating between multiple odorants from the electroantennogram (EAG) signal of a single antenna of an insect is proposed. An antenna of an adult male silkmoth is employed; the silkmoth antenna exhibits an electric potential change when detecting an odor, but the difference in the EAG amplitude and recovery time depends on the type of odorant. Since EAG is a type of neural signal, it is difficult to perform odor discrimination by simply setting the threshold, because it drifts due to noise. Therefore, the Hodgkin-Huxley model (HH model) was applied to the raw EAG signal as a dynamic filter, and the spike firing rate of the output value was calculated using the HH model. Then, the odorants were discriminated against based on the magnitude of the spike firing rate. A series of signal processing was implemented in a microcontroller, and the experiments indicated that all antennae demonstrated a discrimination performance of 90$\%$ or more.

Regulation of olfactory-based sex behaviors in the silkworm by genes in the sex-determination cascade.

Insect courtship and mating depend on integration of olfactory, visual, and tactile cues. Compared to other insects, Bombyx mori, the domesticated silkworm, has relatively simple sexual behaviors as it cannot fly. Here by using CRISPR/Cas9 and electrophysiological techniques we found that courtship and mating behaviors are regulated in male silk moths by mutating genes in the sex determination cascade belonging to two conserved pathways. Loss of Bmdsx gene expression significantly reduced the peripheral perception of the major pheromone component bombykol by reducing expression of the product of the BmOR1 gene which completely blocked courtship in adult males. Interestingly, we found that mating behavior was regulated independently by another sexual differentiation gene, Bmfru. Loss of Bmfru completely blocked mating, but males displayed normal courtship behavior. Lack of Bmfru expression significantly reduced the perception of the minor pheromone component bombykal due to the down regulation of BmOR3 expression; further, functional analysis revealed that loss of the product of BmOR3 played a key role in terminating male mating behavior. Our results suggest that Bmdsx and Bmfru are at the base of the two primary pathways that regulate olfactory-based sexual behavior.

Pore-ridge nanostructures on the surface of trichoid sensilla of the male silkmoth Bombyx mori: Aerodynamic trapping and transporting of the pheromone molecules

This paper tries to reveal the mechanism of the high-efficient adsorption of the sex pheromone by the trichoid sensilla of the male silk moth Bombyx mori. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to acquire the topographies and nanostructures of the surfaces of the trichoid sensilla. SEM and AFM images present mostly regular pore-ridge nanostructures on the sensilla, and all the pores are located at or near the feet of the ridges. AFM phase-shift images demonstrate that the variation of phase-shift, which appears along the ridge cannot simply be attributed to heterogeneity in surface lipid properties, for the phase-shift was present in the same region with the sudden difference in height. Simulations of computational fluid dynamics were applied to investigate the effects on the airflow velocity field and streamlines by the pore-ridge nanostructures and the antenna vibration. Simulation results indicate that the airflow vortexes that form on the sensillum surface are generated by the combined effect of ambient airflow and pore-ridge structure as well as spontaneous vibration of the antenna. We suggest that the vortex intercepts and traps the pheromone molecules passing nearby, and transports them through its periodical movement to the pore. We speculate that the vortex is the aerodynamic factor benefitting the highly efficient adsorption of pheromone molecules.

Mapping of Courtship Behavior-Induced Neural Activity in the Thoracic Ganglia of Silkmoth Bombyx mori by an Immediate Early Gene, Hr38.

In the central nervous system of insects, motor patterns are generated in the thoracic ganglia under the control of brain, where sensory information is integrated and behavioral decisions are made. Previously, we established neural activity-mapping methods using an immediate early gene, BmHr38, as a neural activity marker in the brain of male silkmoth Bombyx mori. In the present study, to gain insights into neural mechanisms of motor-pattern generation in the thoracic ganglia, we investigated expression of BmHr38 in response to sex pheromone-induced courtship behavior. Levels of BmHr38 expression were strongly correlated between the brain and thoracic ganglia, suggesting that neural activity in the thoracic ganglia is tightly controlled by the brain. In situ hybridization of BmHr38 revealed that 20-30% of thoracic neurons are activated by courtship behavior. Using serial sections, we constructed a comprehensive map of courtship behaviorinduced activity in the thoracic ganglia. These results provide important clues into how complex courtship behavior is generated in the neural circuits of thoracic ganglia.

Insect-controlled Robot: A Mobile Robot Platform to Evaluate the Odor-tracking Capability of an Insect.

Robotic odor source localization has been a challenging area and one to which biological knowledge has been expected to contribute, as finding odor sources is an essential task for organism survival. Insects are well-studied organisms with regard to odor tracking, and their behavioral strategies have been applied to mobile robots for evaluation. This "bottom-up" approach is a fundamental way to develop biomimetic robots; however, the biological analyses and the modeling of behavioral mechanisms are still ongoing. Therefore, it is still unknown how such a biological system actually works as the controller of a robotic platform. To answer this question, we have developed an insect-controlled robot in which a male adult silkmoth (Bombyx mori) drives a robot car in response to odor stimuli; this can be regarded as a prototype of a future insect-mimetic robot. In the cockpit of the robot, a tethered silkmoth walked on an air-supported ball and an optical sensor measured the ball rotations. These rotations were translated into the movement of the two-wheeled robot. The advantage of this "hybrid" approach is that experimenters can manipulate any parameter of the robot, which enables the evaluation of the odor-tracking capability of insects and provides useful suggestions for robotic odor-tracking. Furthermore, these manipulations are non-invasive ways to alter the sensory-motor relationship of a pilot insect and will be a useful technique for understanding adaptive behaviors.

Antioxidant Activity and Stability Study of Peptides from Enzymatically Hydrolyzed Male Silkmoth

The antioxidant activity and stability of peptides from male silkmoth hydrolysates (MSH) was studied as antioxidant ingredients in potential food applications. MSH were isolated into five fractions: MSH-I (MW > 30 kDa), MSH-II (10–30 kDa), MSH-III (5–10 kDa), MSH-IV (3–5 kDa), and MSH-V (<3 kDa) by ultrafiltration. The antioxidant activity, molecular weight distribution and amino acid contents of each fraction were evaluated. The effects of NaCl, temperature, pH, light intensity and simulated gastrointestinal digestion were assessed to determine the stability of peptides. The antioxidant capacity of MSH-I as a major antioxidant component was 1,950 µmol/g in ORAC assay and 275 µmol/g in DPPH scavenging method. MSH showed good stability when salinity was less than 4%, temperature below 60C, and pH between 4 and 8. The antioxidant activity could retain about 80% after simulated gastrointestinal digestion. In conclusion, the MSH is a promising resource for stable antioxidant peptides applied in functional food processing and storage. Practical Applications In sericulture, the total cocoon yield has been 738,100 tons, the silk yield has been 93,000 tons, even about 60,000 tons of tussah moth cocoons produced in China in 2005. However, accompanied by the ascension of the silk consumpution, the sonsiderable amount of by-product and sericulture waste presented rising trend yearly. While, moth as insect having allergy protein, which use has been limited, but it has an excellent protein and active peptides and other ingredients (to improve immunity and sexual function), comprehensive utilization of silkmoth turns into meaningful topics. Using enzyme technology or fermentation technology (Protein degradation) to develop its protein resource utilization in silkmoth becomes the effective tools to solve this problem. The present experiments studied antioxidant activity by ultrafiltration technology and stability study of peptides from enzymatically hydrolyzed male silkmoth.

Male-silkmoth-inspired routing algorithm for large-scale wireless mesh networks

This paper proposes an insect behavior-inspired routing algorithm for large-scale wireless mesh networks. The proposed algorithm is adapted from the behavior of an insect called Bombyx mori, a male silkmoth. Its unique behavior is its flying technique to find the source of pheromones. The algorithm consists of two steps: the shortest-path algorithm and the zigzag-path algorithm. First, the shortest-path algorithm is employed to transmit data. After half of the total hops, the zigzag-path algorithm, which is based on the movement of the male B. mori, is applied. In order to adapt the biological behavior to large-scale wireless mesh networks, we use a mesh topology for implementing the algorithm. Simulation results show that the total energy used and the decision time for routing of the proposed algorithm are improved under certain conditions.

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.

A simple behaviour provides accuracy and flexibility in odour plume tracking—the robotic control of sensory-motor coupling in silkmoths

Odour plume tracking is an essential behaviour for animal survival. A fundamental strategy for this is to move upstream and then across-stream. Male silkmoths, Bombyx mori, display this strategy as a pre-programmed sequential behaviour. They walk forward (surge) in response to the female sex pheromone and perform a zigzagging 'mating dance'. Though pre-programmed, the surge direction is modulated by bilateral olfactory input and optic flow. However, the nature of the interaction between these two sensory modalities and contribution of the resultant motor command to localizing an odour source are still unknown. We evaluated the ability of the silkmoth to localize an odour source under conditions of disturbed sensory-motor coupling, using a silkmoth-driven mobile robot. The significance of the bilateral olfaction of the moth was confirmed by inverting the olfactory input to the antennae, or its motor output. Inversion of the motor output induced consecutive circling, which was inhibited by covering the visual field of the moth. This suggests that the corollary discharge from the motor command and the reafference of self-generated optic flow generate compensatory signals to guide the surge accurately. Additionally, after inverting the olfactory input, the robot successfully tracked the odour plume by using a combination of behaviours. These results indicate that accurate guidance of the reflexive surge by integrating bilateral olfactory and visual information with innate pre-programmed behaviours increases the flexibility to track an odour plume even under disturbed circumstances.

Establishment of tools for neurogenetic analysis of sexual behavior in the silkmoth, Bombyx mori.

BackgroundSilkmoth, Bombyx mori, is an ideal model insect for investigating the neural mechanisms underlying sex pheromone-induced innate behavior. Although transgenic techniques and the GAL4/UAS system are well established in the silkmoth, genetic tools useful for investigating brain function at the neural circuit level have been lacking.ResultsIn the present study, we established silkmoth strains in which we could visualize neural projections (UAS-mCD8GFP) and cell nucleus positions (UAS-GFP.nls), and manipulate neural excitability by thermal stimulation (UAS-dTrpA1). In these strains, neural projections and nucleus position were reliably labeled with green fluorescent protein in a GAL4-dependent manner. Further, the behavior of silkworm larvae and adults could be controlled by GAL4-dependent misexpression of dTrpA1. Ubiquitous dTrpA1 misexpression led both silkmoth larvae and adults to exhibit seizure-like phenotypes in a heat stimulation-dependent manner. Furthermore, dTrpA1 misexpression in the sex pheromone receptor neurons of male silkmoths allowed us to control male sexual behavior by changing the temperature. Thermally stimulated male silkmoths exhibited full sexual behavior, including wing-flapping, orientation, and attempted copulation, and precisely approached a thermal source in a manner similar to male silkmoths stimulated with the sex pheromone.ConclusionThese findings indicate that a thermogenetic approach using dTrpA1 is feasible in Lepidopteran insects and thermogenetic analysis of innate behavior is applicable in the silkmoth. These tools are essential for elucidating the relationships between neural circuits and function using neurogenetic methods.

Dynamic use of optic flow during pheromone tracking by the male silkmoth, Bombyx mori.

Several insects require both olfactory and visual cues during odour-source localisation to successfully locate an odour source. In the male silkmoth, Bombyx mori, detection of the female sex pheromone triggers a programmed walking pattern, starting from a surge (straight-line walking) followed by zigzag walking. Although pheromone-triggered behaviour in silkmoths is well understood, the role of visual cues remains obscure. To address this question, we performed behavioural experiments on tethered-walking moths by recording their locomotion during stimulation with a pheromone and a visual motion pattern (optic flow). The experiments were conducted under open- and closed-loop visual stimuli. We found that the use of optic flow input was determined by the behavioural state of surge and zigzagging. Silkmoths exhibited an optomotor response, which is a behavioural visual response, by turning towards the same direction as optic flow stimuli only during surge, but not during zigzagging. In addition, modulation of the zigzag walking pattern was observed when the moths were presented with biased closed-loop visual stimuli (visual feedback with biased constant optic flow); however, the directional preference mechanism was different from that of the optomotor response. Based on these findings, we suggest that the optomotor response is utilised for course control during straight-line walking, whereas the absence of optomotor response during zigzagging is used to effectively perform the programmed walking pattern. Considering the neural basis of programmed behaviour, we speculate that at least two visual pathways are involved in the state-dependent use of optic flow during odour tracking behaviour in silkmoths.

Silkmoths alternate optomotor response

![][1] Once a male silkmoth picks up the whiff of a female, very little will deter him from pursuit. Focusing his full attention on the task, the flightless male doggedly walks (surges) along her tantalising pheromone trail, switching to various zigzagging motions when he slips out of

Antenna-Specific Glutathione S-Transferase in Male Silkmoth Bombyx mori

Glutathione S-transferases (GSTs) are multifunctional enzymes that are widely distributed in different species. GSTs detoxify exogenous and endogenous substances by conjugation to reduced glutathione. We characterized BmGSTD4, an antenna-specific GST, in male silkmoths. The full-length mRNA of Bmgstd4 was cloned by RACE-PCR and contained an open reading frame of 738 bp encoding a 245 amino acid protein. The antenna specificity of BmGSTD4 was validated at the mRNA and protein levels and BmGSTD4 was shown to localize in the sensillum of male silkmoth antennae. Homology modeling and multi-sequence alignment suggested that BmGSTD4 was a typical GST belonging to the δ class and had a canonical GST fold with a conserved N-terminus, including a glutathione-binding site and a C-terminal domain harboring a hydrophobic substrate-binding site. Restricted expression of BmGSTD4 in silkmoth antennae combined with GST activity suggested that BmGSTD4 was involved in the detoxification of harmful chemicals.

Visualization of Neural Activity in Insect Brains Using a Conserved Immediate Early Gene, Hr38

Many insects exhibit stereotypic instinctive behavior [1-3], but the underlying neural mechanisms are not well understood due to difficulties in detecting brain activity in freely moving animals. Immediate early genes (IEGs), such as c-fos, whose expression is transiently and rapidly upregulated upon neural activity, are powerful tools for detecting behavior-related neural activity in vertebrates [4, 5]. In insects, however, this powerful approach has not been realized because no conserved IEGs have been identified. Here, we identified Hr38 as a novel IEG that is transiently expressed in the male silkmoth Bombyx mori by female odor stimulation. Using Hr38 expression as an indicator of neural activity, we mapped comprehensive activity patterns of the silkmoth brain in response to female sex pheromones. We found that Hr38 can also be used as a neural activity markerin the fly Drosophila melanogaster. Using Hr38, we constructed a neural activity map of the fly brain that partiallyoverlaps with fruitless (fru)-expressing neurons in response to female stimulation. These findings indicate that Hr38 is a novel and conserved insect neural activity marker gene that will be useful for a wide variety of neuroethologic studies.