Analysis of Multisensory-Motor Integration in Olfactory Navigation of Silkmoth, Bombyx mori, using Virtual Reality System

Most animals survive and thrive due to navigation behavior to reach their destinations. In order to navigate, it is important for animals to integrate information obtained from multisensory inputs and use that information to modulate their behavior. In this study, by using a virtual reality (VR) system for an insect, we investigated how an adult silkmoth integrates visual and wind direction information during female search behavior (olfactory behavior). According to the behavioral experiments using the VR system, the silkmoth had the highest navigation success rate when odor, vision, and wind information were correctly provided. However, we found that the success rate of the search significantly reduced if wind direction information was provided that was incorrect from the direction actually detected. This indicates that it is important to acquire not only odor information, but also wind direction information correctly. In other words, Behavior was modulated by the degree of co-incidence between the direction of arrival of the odor and the direction of arrival of the wind, and posture control (angular velocity control) was modulated by visual information. We mathematically modeled the modulation of behavior using multisensory information and evaluated it by simulation. As a result, the mathematical model not only succeeded in reproducing the actual female search behavior of the silkmoth, but can also improve search success relative to the conventional odor source search algorithm.


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In many organisms, including humans, appropriate behavior is determined based on the integra-29 tion of different kinds of information from the environment. Examples of information obtained Odor Wind Vision Figure 1. The virtual reality (VR) system for olfactory navigation of the insect and a list of experimental conditions. A: The VR system is equipped with a stimulator of odor, vision, and wind, and is connected to a virtual odor field. The insect on the VR device performs olfactory navigation in virtual space. B: Definition of presentation way of each sensory stimulus. C: The odor is presented under all conditions. "•", "×", and "•" indicate presented, not presented, and presented from the opposite direction to the actual direction, respectively.  to right. The search performance of the silkmoth using the constructed VR system was the same as that in free-walking experiments (see Supplemental Materials). We carried out experiments using 91 the VR by changing the number of sensory inputs as follows (Fig. 1C);

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"•" indicates that we presented the environmental information to the silkmoth, and "×" indi-95 cates that we did not present the environmental information to the silkmoth (Fig. 1C). "•" indicates 96 that the stimulus was presented from the opposite direction. For example, "Inverse wind" means 97 that the virtual insect in the computer received wind from the front, while the real silkmoth received 98 wind from the back. Three repetitions of the experiment were conducted using 10 silkworm moths 99 for each environmental condition ( =30). We set a time limit of 300 seconds because theoretically, 100 the silkmoth could reach the odor source in an infinite time. The search was considered a failure 101 if the moth did not enter a radius of 10 mm from the odor source within the time limit.

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Wind and visual effects on the olfactory navigation 103 We compared the search performance in response to different types of stimuli by presenting wind 104 and visual stimuli in addition to olfactory stimulus in all conditions. We first measured the behavior 105 when wind or visual stimuli is providing in addition to odor stimulus (Group 1). We created a migra-106 tion probability map in order to visualize the effects of differences in environmental conditions on 107 the behavioral trajectory ( Fig. 2A-E). To quantitatively evaluate the similarity between the migra-108 tion probability maps, we calculated the earth mover's distance (EMD) (Rubner et al., 1997 Cond. 5 Figure 2. The result of visually expressing the trajectory under each experimental condition with a migration probability map (A-E). The white area in the figure indicates that the moth did not move in that space. A quantitative evaluation using earth mover's distance, EMD is illustrated in F. F was the result of calculating the similarity (EMD) based on the trajectory of cond. 1 (odor presentation only). The lower the value, the higher the similarity, and the higher the value, the lower the similarity of the trajectories.  In the previous section, it was found that wind direction information, in addition to odor informa-139 tion, contributed to improving the success rate in searching for the odor source. Here, we analyze 140 in detail how behavior is modulated by visual and wind information using three experimental con-141 ditions: a forward condition (cond. i), an inverse condition for wind direction information (cond. ii), 142 and an inverse condition for visual stimuli (cond. iii). 143 We first analyzed the effect of wind direction information on odor source search behavior. The 144 results of calculating EMD in the previous section suggested that the search behavior was signif-

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icantly affected when wind direction information was presented, in addition to odor information.

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Because the key stimulus for female searching behavior in silkmoths is odor, and because previ-147 ous studies (e.g. (Kikas et al., 2001)) have reported that odor detection frequency is related to the 148 distance from the odor source, and that odor detection frequency modulates behavior, we ana-149 lyzed odor as a state variable. Here, odor detection frequency is defined as the frequency at which  Changes in translational velocity and angular velocity when the wind was presented from the direction opposite to the actual direction (cond. ii). The translational velocity was constant regardless of the odor detection frequency, and the peak position of the angular velocity was 0.5 times that of cond. i. C: Comparison of angular velocities when the visual information was presented correctly (cond. i) and when it was presented in the opposite direction (cond. iii). Vision was used to equalize the speed of the left-right rotation.

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odor and wind detection coincided.
Here, we investigated how behavioral modulation obtained from behavioral experiments using a

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VR system contributes to the odor source search. A silkmoth moves by walking on a two-dimensional 192 plane with six legs, but it does not move in the lateral direction. Therefore, we assumed that it has (1) By passing through the directional discriminator and frequency counter, the odor is converted 215 into information such as whether it was detected by the left or right antennae and how much odor 216 it was exposed to. The wind is converted into wind direction information. A comparator is used 217 9 of 18 to determine whether the direction of odor detection and the direction of the wind are the same, and the results are input to the straight-ahead speed and angular velocity controllers, which also 219 receive odor detection frequency. Visual information controls the angular velocity of the left and 220 right rotational movements following which olfactory behavior takes place. From this information, 221 the movement speed output is calculated, and the behavior is generated. We compared the MiM2 222 algorithm to the previous surge-zigzagging algorithm and the casting algorithm, which uses wind 223 information for searching in a moth-inspired algorithm proposed in a previous study (Li et al., . 225 The simulation environment employed a virtual environment similar to that used in the behav-   oped for visual-dominated navigation, it was difficult to measure olfactory-dominated navigation, 267 which was the focus of our research. Therefore, we developed a novel multimodal VR system that 268 allowed us to measure changes in navigation behavior when the olfactory, visual, and wind direc-269 tions of the silkmoth were modified. Conventionally, previous studies on the silkmoth have shown 270 that (1) a "mating dance" was elicited in response to sexual pheromones (OBARA, 1979), (2) the con-271 dition in which visual stimuli was presented immediately after the reception of sex pheromones 272 influenced the subsequent rotational behavior (Pansopha et al., 2014), and (3) behavioral inhibition 273 occurred in response to frontal winds (Shigaki et al., 2019a). However, the integration of the above 274 phenomena during navigation has not been investigated. By presenting three types of sensory in-275 formation simultaneously as well as continuously, we were able to clarify the roles of each type of 276 sensory information in navigation. Moreover, mathematical modeling and simulation showed that 277 multimodal sensory information improved silkmoth navigation.  et al., 2020). If we assume that the "tempo" is the rate of odor de-299 tection per unit time, it is related to the cycle in which the odor arrives. We hypothesized that the 300 silkmoth, like flying insects such as drosophila, modulates its behavior based on the "tempo" of the 301 odor, therefore we included odor frequency in our analyses.

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When the direction of the wind and the odor coincided, movement speed peaked when the 303 odor frequency was 0.7-0.8, which is similar to the frequency at which a female silkmoth releases 304 sex pheromones (0.79 ± 0.05 Hz) (Fujiwara et al., 2014). Based on these findings, we can hypothe- . 315 In an earlier study investigating the direction of wind and odor in the environment, the direc-316 12 of 18 tion of wind and odor were the same in the open field experiment (no obstacles) (Murlis et al.,  2000). However, the direction of wind and odor is not always the same in a complex environment 318 such as a forest with many trees (Murlis et al., 2000). Our data showed that in situations where 319 the wind and odor direction do not match, the silkmoth always moves at the same rate given any 320 odor detection frequency. This may be a chemical tracking strategy to avoid leaving the odor range 321 by moving at a speed lower than normal speed, and suggests that the silkmoth is estimating the 322 degree of environmental turbulence and modulating its behavior based on the odor and wind di-323 rection information. This strategy may be applicable to an engineering search system that switches  (Demir et al., 2020). In this study, we found that behavioral 346 modulation occurred based on the relationship between the direction of the odor arrival and the 347 wind, and we used our data to reproduce a behavioral trajectory that was not only better than that 348 of the conventional bio-inspired algorithm, but was also more similar to the search behavior of the 349 actual silkmoth. Therefore, it is clear that a probabilistic and time-varying behavior modulation 350 mechanism has a better search performance than a time-invariant search algorithm.

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Adult male moths were cooled at 16 • C one day after eclosion to reduce their activity, and were 355 tested within 2-7 days after eclosion. Before the experiments, the moths were kept at room tem-356 perature (25-28 • C) for at least 10 min.

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Virtual odor field 358 To generate a virtual odor field closely mimicking reality, smoke was emitted into the actual en- to extract only smoke with simple thresholding because the noise caused by dust has the same 380 luminance as does smoke. Therefore, we adopted a method that focuses on connected compo-381 nents to remove noise while maintaining the shape of the smoke. In grouping based on connected 382 components, two objects are considered connected if adjacent pixels in a binary image take the 383 value of 1. Because smoke exists as a mass, it can be inferred that it has an area above a certain 384 level when divided into connected components. For this reason, we removed the pixels whose 385 connected components were not in adjacent pixels and whose area did not exceed a certain level.

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These processes were performed with source code using OpenCV.

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The behavioral experiment was carried out using a homemade virtual reality system (a photograph 389 of the actual device is shown in Fig. 1A). The traditional tethered measurement system is used for 390 behavior measurement, and a stimulator that presents odor, visual, and wind direction informa-391 tion is installed around the tethered measurement system (Figure 1-Figure supplement 1A). The 392 details of each stimulator are as follows.

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Odor stimulator 394 We provided sex pheromone stimulation to both antennae of a silkmoth using Bombykol ((E,Z)-
We applied the equation to all trial trajectories and summarized them, and then calculated the For all data analyses, R version 4.0.3 was used (R Core Team). All earth mover's distance calculations 453 were performed using the python 3.7 language.

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Odor source search algorithm 455 The details of the three algorithms for which simulation experiments were performed are de-456 scribed here.   Data comparison between free walking experiment and VR experiment. A: VR system configuration diagram. Two microcomputers were used to control the stimulator and measure the amount of rotation of the sphere. B-D: Evaluation experiment results of each stimulator. B is a graph showing the change in the heading angle of the silkmoth. It was confirmed that when the odor was presented from the upper part of the antennae, the female search behavior was elicited while changing the heading angle significantly. C investigated whether the visual motion reflex was elicited by the visual stimulator by observing the change in the angle of the neck. Because the neck tilt was the largest in the same speed band as the angular velocity of the silk moth, it was confirmed that the visual stimulus could be presented correctly. D is a snapshot of the push-pull rectifier, which is a wind stimulator, visualized by PIV. Since the yellow line represents the streamline and does not generate vortices, it was confirmed that rectification can be generated by push-pull. E: Comparison of search success rates between free walking experiments and VR experiments (Fisher's exact test, < 0.05). The free walking experiment is the result of two repeated experiments using 15 silkmoths ( = 30). The environment of the free walking experiment was set to be the same as the virtual environment of the VR system. F: Comparison of a relative length. The relative length is an evaluation value that standardizes the distance actually traveled by the shortest distance to the odor source. This makes it possible to evaluate how much action was taken in response to sensory input. Because there was no difference between free walking and VR (Welch's t-test, < 0.05), it is highly possible that the search behavior expressed by a silkmoth in the VR experiment is the same as the behavior in the free walking experiment.