Connectome of the lamina reveals the circuit for early color processing in the visual pathway of a butterfly.

Connectomics has become a standard neuroscience methodology in a few model animals,1 with the visual system being a popular target of study.2-5 Combining connectomics with circuit and behavioral physiology, recent studies on the color vision of the fruit fly Drosophila melanogaster have focused on the mechanisms underlying early wavelength processing in the optic ganglia.6-8 However, the color vision capabilities of D. melanogaster are limited,9 compared with many flower-visiting insects.10,11 For example, a butterfly Papilio xuthus has six spectral classes of photoreceptors. Each ommatidium contains nine photoreceptors in one of three fixed combinations, making the eye an array of three spectrally distinct ommatidia types.12 Behaviorally, P. xuthus can detect 1 nm differences in light wavelength across the spectrum from ultraviolet to red, outperforming humans.13 What is the neuronal basis of such precise color vision? How does such a system evolve? Addressing these questions requires comparative studies at the circuit level. Here, we performed a connectome analysis in the first optic ganglion, the lamina, of P. xuthus. The lamina comprises cartridges, each typically containing nine photoreceptor axons from a single ommatidium and four second-order neurons. We found abundant inter-photoreceptor connections, which are absent in the lamina of D. melanogaster. We also identified connections between neighboring cartridges, particularly those receiving inputs from spectrally distinct ommatidia. The linear summation of synaptic connections well explains the spectral sensitivity of photoreceptors and second-order neurons in the lamina.

Cortical-like colour-encoding neurons in the mushroom body of a butterfly.

Colour is an important visual modality for many animals including insects. The flower-foraging swallowtail butterfly Papilio xuthus has spectrally acute chromatic vision using UV-, blue-, green- and red-sensitive photoreceptors1. The spectral organization of Papilio's retina is well understood but, as for other insects, how chromatic information is processed in higher order brain regions remains unclear. To identify neurons underlying color perception in Papilio, we have investigated the spectral properties of the visual inputs to the mushroom body (MB), a brain region implicated in learning and memory. By recording intracellular responses to a series of monochromatic lights, combined with dye injection, we have revealed a wide variety of spectral responses in three morphologically distinct neuron types. These heterogeneous responses are characterized by colour opponency and sharp tuning to particular wavelengths, which do not in general align with the sensitivities of the retinal photoreceptors, and presumably contribute to Papilio's acute wavelength discrimination. This finding provides new insights into the processing underlying insect colour vision.

Swallowtail Butterflies Use Multiple Visual Cues to Select Oviposition Sites.

Flower-foraging Japanese yellow swallowtail butterflies, Papilio xuthus, exhibit sophisticated visual abilities. When ovipositing, females presumably attempt to select suitable leaves to support the growth of their larval offspring. We first established that butterflies indeed select particular leaves on which to lay eggs; when presented with a single Citrus tree, butterflies significantly favored two out of 102 leaves for oviposition. These preferences were observed across many individuals, implying that they were not merely idiosyncratic, but rather based on properties of the leaves in question. Because the butterflies descended towards the leaves rather directly from a distance, we hypothesized that they base their selection on visual cues. We measured five morphological properties (height, orientation, flatness, roundness, and size) and four reflective features (green reflectance, brightness, and degree and angle of linear polarization). We found that the number of eggs laid upon a leaf was positively correlated with its height, flatness, green reflectance, and brightness, and negatively correlated with its degree of polarization, indicating that these features may serve as cues for leaf selection. Considering that other studies report ovipositing butterflies' preference for green color and horizontally polarized light, butterflies likely use multiple visual features to select egg-laying sites on the host plant.

Retinal organization and visual abilities for flower foraging in swallowtail butterflies.

Papilio butterflies' ability to forage for flowers relies upon multiple visual cues such as color, brightness, and motion. Papilio learns the color of rewarding flowers and detects it at a distance. Its color vision is based on four photoreceptor classes: UV, blue, green, and red, providing sensitive wavelength discrimination. These four receptor classes also contribute to the perception of brightness and polarization. Papilio's motion vision is based on a different set of receptors: green, red, and broad band. This implies that two visual pathways exist in Papilio. The contribution of several receptor classes not only for chromatic vision but also achromatic vision likely enhances the butterfly's ability to detect flowers in complex visual environments.

Monopolatic motion vision in the butterfly Papilio xuthus.

The swallowtail butterfly Papilio xuthus can perceive the linear polarization of light. Using a novel polarization projection system, we recently demonstrated that P. xuthus can detect visual motion based on polarization contrast. In the present study, we attempt to infer via behavioural experiments the mechanism underlying this polarization-based motion vision. Papilio xuthus do not perceive contrast between unpolarized and diagonally polarized light, implying that they cannot unambiguously estimate angle and degree of polarization, at least as far as motion detection is concerned. Furthermore, they conflate brightness and polarization cues, such that bright vertically polarized light resembles dim unpolarized light. These observations are consistent with a one-channel 'monopolatic' detector mechanism. We extend our existing model of motion vision in P. xuthus to incorporate these polarization findings, and conclude that the photoreceptors likely to form the basis for the putative monopolatic polarization detector are R3 and R4, which respond maximally to horizontally polarized green light. R5-R8, we propose, form a polarization-insensitive secondary channel tuned to longer wavelengths of light. Consistent with this account, we see greater sensitivity to polarization for green-light stimuli than for subjectively equiluminant red ones. Somewhat counter-intuitively, our model predicts greatest sensitivity to vertically polarized light; owing to the non-linearity of photoreceptor responses, light polarized to an angle orthogonal to a monopolatic detector's orientation offers the greatest contrast with unpolarized light.

The Dominant Role of Visual Motion Cues in Bumblebee Flight Control Revealed Through Virtual Reality.

Flying bees make extensive use of optic flow: the apparent motion in the visual scene generated by their own movement. Much of what is known about bees' visually-guided flight comes from experiments employing real physical objects, which constrains the types of cues that can be presented. Here we implement a virtual reality system allowing us to create the visual illusion of objects in 3D space. We trained bumblebees, Bombus ignitus, to feed from a static target displayed on the floor of a flight arena, and then observed their responses to various interposing virtual objects. When a virtual floor was presented above the physical floor, bees were reluctant to descend through it, indicating that they perceived the virtual floor as a real surface. To reach a target at ground level, they flew through a hole in a virtual surface above the ground, and around an elevated virtual platform, despite receiving no reward for avoiding the virtual obstacles. These behaviors persisted even when the target was made (unrealistically) visible through the obstructing object. Finally, we challenged the bees with physically impossible ambiguous stimuli, which give conflicting motion and occlusion cues. In such cases, they behaved in accordance with the motion information, seemingly ignoring occlusion.

A Novel Display System Reveals Anisotropic Polarization Perception in the Motion Vision of the Butterfly Papilio xuthus.

While the linear polarization of light is virtually invisible to humans, many invertebrates' eyes can detect it. How this information is processed in the nervous system, and what behavioral function it serves, are in many cases unclear. One reason for this is the technical difficulty involved in presenting images or video containing polarization contrast, particularly if intensity and/or color contrast is also required. In this primarily methods-focused article, we present a novel technique based on projecting video through a synchronously rotating linear polarizer. This approach allows the intensity, angle of polarization, degree of linear polarization, and potentially also color of individual pixels to be controlled independently. We characterize the performance of our system, and then use it to investigate the relationship between polarization and motion vision in the swallowtail butterfly Papilio xuthus. Although this animal has photoreceptors sensitive to four different polarization angles, we find that its motion vision cannot distinguish between diagonally-polarized and unpolarized light. Furthermore, it responds more strongly to vertically-polarized moving objects than horizontally-polarized ones. This implies that Papilio's polarization-based motion detection employs either an unbalanced two-channel (dipolatic) opponent architecture, or possibly a single-channel (monopolatic) scheme without opponent mechanisms.

Red-shift of spectral sensitivity due to screening pigment migration in the eyes of a moth, Adoxophyes orana.

BACKGROUND: We have found that the spectral sensitivity of the compound eye in the summer fruit tortrix moth (Adoxophyes orana) differs in laboratory strains originating from different regions of Japan. We have investigated the mechanisms underlying this anomalous spectral sensitivity. METHODS: We applied electrophysiology, light and electron microscopy, opsin gene cloning, mathematical modeling, and behavioral analysis. RESULTS: The ERG-determined spectral sensitivity of dark-adapted individuals of all strains peaks around 520 nm. When light-adapted, the spectral sensitivity of the Nagano strain narrows and its peak shifts to 580 nm, while that in other strains remains unchanged. All tested strains appear to be identical in terms of the basic structure of the eye, the pigment migration in response to light- and dark-adaptation, and the molecular structure of long-wavelength absorbing visual pigments. However, the color of the perirhabdomal pigment clearly differs; it is orange in the Nagano strain and purple in the others. The action spectrum of phototaxis appears to be shifted towards longer wavelengths in the Nagano individuals. CONCLUSIONS: The spectral sensitivities of light-adapted eyes can be modeled under the assumption that this screening pigment plays a crucial role in determining the spectral sensitivity. The action spectrum of phototaxis indicates that the change in the eye spectral sensitivity is behaviorally relevant.

Multisensory integration in Lepidoptera: Insights into flower-visitor interactions.

As most work on flower foraging focuses on bees, studying Lepidoptera can offer fresh perspectives on how sensory capabilities shape the interaction between flowers and insects. Through a combination of innate preferences and learning, many Lepidoptera persistently visit particular flower species. Butterflies tend to rely on their highly developed sense of colour to locate rewarding flowers, while moths have evolved sophisticated olfactory systems towards the same end. However, these modalities can interact in complex ways; for instance, butterflies' colour preference can shift depending on olfactory context. The mechanisms by which such cross-modal interaction occurs are poorly understood, but the mushroom bodies appear to play a central role. Because of the diversity seen within Lepidoptera in terms of their sensory capabilities and the nature of their relationships with flowers, they represent a fruitful avenue for comparative studies to shed light on the co-evolution of flowers and flower-visiting insects.

The butterfly Papilio xuthus detects visual motion using chromatic contrast.

Many insects' motion vision is achromatic and thus dependent on brightness rather than on colour contrast. We investigate whether this is true of the butterfly Papilio xuthus, an animal noted for its complex retinal organization, by measuring head movements of restrained animals in response to moving two-colour patterns. Responses were never eliminated across a range of relative colour intensities, indicating that motion can be detected through chromatic contrast in the absence of luminance contrast. Furthermore, we identify an interaction between colour and contrast polarity in sensitivity to achromatic patterns, suggesting that ON and OFF contrasts are processed by two channels with different spectral sensitivities. We propose a model of the motion detection process in the retina/lamina based on these observations.

The roles of visual parallax and edge attraction in the foraging behaviour of the butterfly Papilio xuthus.

Several examples of insects using visual motion to measure distance have been documented, from locusts peering to gauge the proximity of prey, to honeybees performing visual odometry en route between the hive and a flower patch. However, whether the use of parallax information is confined to specialised behaviours like these or represents a more general purpose sensory capability, is an open question. We investigate this issue in the foraging swallowtail butterfly Papilio xuthus, which we trained to associate a target presented on a monitor with a food reward. We then tracked the animal's flight in real-time, allowing us to manipulate the size and/or position of the target in a closed-loop manner to create the illusion that it is situated either above or below the monitor surface. Butterflies are less attracted to (i.e. slower to approach) targets that appear, based on motion parallax, to be more distant. Furthermore, we found that the number of abortive descent manoeuvres performed prior to the first successful target approach varies according to the depth of the virtual target, with expansion and parallax cues having effects of opposing polarity. However, we found no evidence that Papilio modulate the kinematic parameters of their descents according to the apparent distance of the target. Thus, we argue that motion parallax is used to identify a proximal target object, but that the subsequent process of approaching it is based on stabilising its edge in the 2D space of the retina, without estimating its distance.

Physiological basis of phototaxis to near-infrared light in Nephotettix cincticeps.

In a previous study of the phototaxis of green rice leafhoppers, Nephotettix cincticeps (Hemiptera, Cicadellidae), we found positive responses to 735 nm light. Here, we investigated the mechanism underlying this sensitivity to near-infrared light. We first measured the action spectrum using a Y-maze with monochromatic lights from 480 to 740 nm. We thus found that the action spectrum peaks at 520 nm in the tested wavelength range, but that a significant effect is still observed at 740 nm, albeit with a sensitivity 5 log units lower than the peak. Second, we measured the spectral sensitivity of the eye, and found that the sensitivity in the long-wavelength region parallels the behaviorally determined action spectrum. We further identified mRNAs encoding opsins of ultraviolet, blue, and green-absorbing visual pigments, and localized the mRNAs in the ommatidia by in situ hybridization. The electrophysiology, molecular biology and the anatomy of the eye together indicate that the eyes of N. cincticeps do not contain true "red" receptors, but rather that the behavioral response to near-infrared light is mediated by the tail sensitivity of the green receptors in the long-wavelength region of the spectrum.

A model of visual-olfactory integration for odour localisation in free-flying fruit flies.

Flying fruit flies (Drosophila melanogaster) locate a concealed appetitive odour source most accurately in environments containing vertical visual contrasts. To investigate how visuomotor and olfactory responses may be integrated, we examine the free-flight behaviour of flies in three visual conditions, with and without food odour present. While odour localisation is facilitated by uniformly distributed vertical contrast as compared with purely horizontal contrast, localised vertical contrast also facilitates odour localisation, but only if the odour source is situated close to it. We implement a model of visuomotor control consisting of three parallel subsystems: an optomotor response stabilising the model fly's yaw orientation; a collision avoidance system to saccade away from looming obstacles; and a speed regulation system. This model reproduces many of the behaviours we observe in flies, including visually mediated 'rebound' turns following saccades. Using recordings of real odour plumes, we simulate the presence of an odorant in the arena, and investigate ways in which the olfactory input could modulate visuomotor control. We reproduce the experimental results by using the change in odour intensity to regulate the sensitivity of collision avoidance, resulting in visually mediated chemokinesis. Additionally, it is necessary to amplify the optomotor response whenever odour is present, increasing the model fly's tendency to steer towards features of the visual environment. We conclude that visual and olfactory responses of Drosophila are not independent, but that relatively simple interaction between these modalities can account for the observed visual dependence of odour source localisation.