Molecular logic behind the three-way stochastic choices that expand butterfly colour vision.

Butterflies rely extensively on colour vision to adapt to the natural world. Most species express a broad range of colour-sensitive Rhodopsin proteins in three types of ommatidia (unit eyes), which are distributed stochastically across the retina. The retinas of Drosophila melanogaster use just two main types, in which fate is controlled by the binary stochastic decision to express the transcription factor Spineless in R7 photoreceptors. We investigated how butterflies instead generate three stochastically distributed ommatidial types, resulting in a more diverse retinal mosaic that provides the basis for additional colour comparisons and an expanded range of colour vision. We show that the Japanese yellow swallowtail (Papilio xuthus, Papilionidae) and the painted lady (Vanessa cardui, Nymphalidae) butterflies have a second R7-like photoreceptor in each ommatidium. Independent stochastic expression of Spineless in each R7-like cell results in expression of a blue-sensitive (Spineless(ON)) or an ultraviolet (UV)-sensitive (Spineless(OFF)) Rhodopsin. In P. xuthus these choices of blue/blue, blue/UV or UV/UV sensitivity in the two R7 cells are coordinated with expression of additional Rhodopsin proteins in the remaining photoreceptors, and together define the three types of ommatidia. Knocking out spineless using CRISPR/Cas9 (refs 5, 6) leads to the loss of the blue-sensitive fate in R7-like cells and transforms retinas into homogeneous fields of UV/UV-type ommatidia, with corresponding changes in other coordinated features of ommatidial type. Hence, the three possible outcomes of Spineless expression define the three ommatidial types in butterflies. This developmental strategy allowed the deployment of an additional red-sensitive Rhodopsin in P. xuthus, allowing for the evolution of expanded colour vision with a greater variety of receptors. This surprisingly simple mechanism that makes use of two binary stochastic decisions coupled with local coordination may prove to be a general means of generating an increased diversity of developmental outcomes.

Spectral organization of the compound eye of a migrating nymphalid, the chestnut tiger butterfly Parantica sita.

Several butterflies of family Nymphalidae perform long-distance migration. Extensive studies of migration in the iconic monarch butterfly Danaus plexippus have revealed that vision plays a crucial role in migratory orientation. Differences in the migratory patterns of butterflies suggest that not all species are exposed to the same visual conditions and yet, little is known about how the visual system varies across migratory species. Here, we used intracellular electrophysiology, dye injection and electron microscopy to assess the spectral and polarization properties of the photoreceptors of a migrating nymphalid, Parantica sita Our findings reveal three spectral classes of photoreceptors including ultraviolet, blue and green receptors. The green receptor class contains three subclasses, which are broad, narrow and double-peaking green receptors. Ultraviolet and blue receptors are sensitive to polarized light parallel to the dorso-ventral axis of the animal, while the variety of green receptors are sensitive to light polarized at 45 deg, 90 deg and 135 deg away from the dorso-ventral axis. The polarization sensitivity ratio is constant across spectral receptor classes at around 1.8. Although P. sita has a typical nymphalid eye with three classes of spectral receptors, subtle differences exist among the eyes of migratory nymphalids, which may be genus specific.

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.

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.

Non-linear amplification of graded voltage signals in the first-order visual interneurons of the butterfly Papilio xuthus.

Lamina monopolar cells (LMCs) are the first-order visual interneurons of insects and crustacea, primarily involved in achromatic vision. Here, we investigated morphological and electrophysiological properties of LMCs in the butterfly Papilio xuthus Using intracellular recording coupled with dye injection, we found two types of LMCs. Cells with roundish terminals near the distal surface of the medulla demonstrating no or small depolarizing spikes were classified as L1/2. Cells with elongated terminals deep in the medulla that showed prominent spiking were classified as L3/4. The majority of LMCs of both types had broad spectral sensitivities, peaking between 480 and 570 nm. Depending on the experimental conditions, spikes varied from small to action potential-like events, with their amplitudes and rates decreasing as stimulus brightness increased. When the eye was stimulated with naturalistic contrast-modulated time series, spikes were reliably triggered by high-contrast components of the stimulus. Spike-triggered average functions showed that spikes emphasize rapid membrane depolarizations. Our results suggest that spikes are mediated by voltage-activated Na+ channels, which are mainly inactivated at rest. Strong local minima in the coherence functions of spiking LMCs indicate that the depolarizing conductance contributes to the amplification of graded responses even when detectable spikes are not evoked. We propose that the information transfer strategies of spiking LMCs change with light intensity. In dim light, both graded voltage signals and large spikes are used together without mutual interference, as a result of separate transmission bandwidths. In bright light, signals are non-linearly amplified by the depolarizing conductance in the absence of detectable spikes.

Extraordinary diversity of visual opsin genes in dragonflies.

Dragonflies are colorful and large-eyed animals strongly dependent on color vision. Here we report an extraordinary large number of opsin genes in dragonflies and their characteristic spatiotemporal expression patterns. Exhaustive transcriptomic and genomic surveys of three dragonflies of the family Libellulidae consistently identified 20 opsin genes, consisting of 4 nonvisual opsin genes and 16 visual opsin genes of 1 UV, 5 short-wavelength (SW), and 10 long-wavelength (LW) type. Comprehensive transcriptomic survey of the other dragonflies representing an additional 10 families also identified as many as 15-33 opsin genes. Molecular phylogenetic analysis revealed dynamic multiplications and losses of the opsin genes in the course of evolution. In contrast to many SW and LW genes expressed in adults, only one SW gene and several LW genes were expressed in larvae, reflecting less visual dependence and LW-skewed light conditions for their lifestyle under water. In this context, notably, the sand-burrowing or pit-dwelling species tended to lack SW gene expression in larvae. In adult visual organs: (i) many SW genes and a few LW genes were expressed in the dorsal region of compound eyes, presumably for processing SW-skewed light from the sky; (ii) a few SW genes and many LW genes were expressed in the ventral region of compound eyes, probably for perceiving terrestrial objects; and (iii) expression of a specific LW gene was associated with ocelli. Our findings suggest that the stage- and region-specific expressions of the diverse opsin genes underlie the behavior, ecology, and adaptation of dragonflies.

Innate preference and learning of colour in the male cotton bollworm moth, Helicoverpa armigera.

We investigated colour discrimination and learning in adult males of the nocturnal cotton bollworm moth, Helicoverpa armigera, under a dim light condition. The naive moths preferred blue and discriminated the innately preferred blue from several shades of grey, indicating that the moths have colour vision. After being trained for 2 days to take nectar at a yellow disc, an innately non-preferred colour, moths learned to select yellow over blue. The choice distribution between yellow and blue changed significantly from that of naive moths. However, the dual-choice distribution of the trained moths was not significantly biased to yellow: the preference for blue is robust. We also tried to train moths to grey, which was not successful. The limited ability to learn colours suggests that H armigera may not strongly rely on colours when searching for flowers in the field, although they have the basic property of colour vision.

Photoreceptor projections and receptive fields in the dorsal rim area and main retina of the locust eye.

In many insect species, photoreceptors of a small dorsal rim area of the eye are specialized for sensitivity to the oscillation plane of polarized skylight and, thus, serve a role in sky compass orientation. To further understand peripheral mechanisms of polarized-light processing in the optic lobe, we have studied the projections of photoreceptors and their receptive fields in the main eye and dorsal rim area of the desert locust, a model system for polarization vision analysis. In both eye regions, one photoreceptor per ommatidium, R7, has a long visual fiber projecting through the lamina to the medulla. Axonal fibers from R7 receptors of the dorsal rim area have short side branches throughout the depth of the dorsal lamina and maintain retinotopic projections to the dorsal medulla following the first optic chiasma. Receptive fields of dorsal rim photoreceptors are considerably larger (average acceptance angle 33°) than those of the main eye (average acceptance angle 2.04°) and, taken together, cover almost the entire sky. The data challenge previous reports of two long visual fibers per ommatidium in the main eye of the locust and provide data for future analysis of peripheral networks underlying polarization opponency in the locust brain.

Difference in dynamic properties of photoreceptors in a butterfly, Papilio xuthus: possible segregation of motion and color processing.

The eyes of the Japanese yellow swallowtail butterfly, Papilio xuthus, contain six spectral classes of photoreceptors, each sensitive either in the ultraviolet, violet, blue, green, red or broadband wavelength regions. The green-sensitive receptors can be divided into two subtypes, distal and proximal. Previous behavioral and anatomical studies have indicated that the distal subtype appears to be involved in motion vision, while the proximal subtype is important for color vision. Here, we studied the dynamic properties of Papilio photoreceptors using light stimulation with randomly modulated intensity and light pulses. Frequency response (gain) of all photoreceptor classes shared a general profile-a broad peak around 10 Hz with a declining slope towards higher frequency range. At 100 Hz, the mean relative gain of the distal green receptors was significantly larger than any other receptor classes, indicating that they are the fastest. Photoreceptor activities under dim light were higher in the ultraviolet and violet receptors, suggesting higher transduction sensitivities. Responses to pulse stimuli also distinguished the green receptors from others by their shorter response latencies. We thus concluded that the distal green receptors carry high frequency information in the visual system of Papilio xuthus.

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.

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.

Plant scents modify innate colour preference in foraging swallowtail butterflies.

Flower-visiting insects exhibit innate preferences for particular colours. A previous study demonstrated that naive Papilio xuthus females prefer yellow and red, whereas males are more attracted to blue. Here, we demonstrate that the innate colour preference can be modified by olfactory stimuli in a sexually dimorphic manner. Naive P. xuthus were presented with four coloured discs: blue, green, yellow and red. The innate colour preference (i.e. the colour first landed on) of the majority of individuals was blue. When scent from essential oils of either orange flower or lily was introduced to the room, females' tendency to select the red disc increased. Scents of lavender and flowering potted Hibiscus rosa-sinensis, however, were less effective. Interestingly, the odour of the non-flowering larval host plant, Citrus unshiu, shifted the preference to green in females. In males, however, all plant scents were less effective than in females, such that blue was always the most favoured colour. These observations indicate that interactions between visual and olfactory cues play a more prominent role in females.

Color and polarization vision in foraging Papilio.

This paper gives an overview of behavioral studies on the color and polarization vision of the Japanese yellow swallowtail butterfly, Papilio xuthus. We focus on indoor experiments on foraging individuals. Butterflies trained to visit a disk of certain color correctly select that color among various other colors and/or shades of gray. Correct selection persists under colored illumination, but is systematically shifted by background colors, indicating color constancy and simultaneous color contrast. While their eyes contain six classes of spectral receptors, their wavelength discrimination performance indicates that their color vision is tetrachromatic. P. xuthus innately prefers brighter targets, but can be trained to select dimmer ones under certain conditions. Butterflies trained to a dark red stimulus select an orange disk presented on a bright gray background over one on dark gray. The former probably appears darker to them, indicating brightness contrast. P. xuthus has a strong innate preference for vertically polarized light, but the selection of polarized light changes depending on the intensity of simultaneously presented unpolarized light. Discrimination of polarization also depends on background intensity. Similarities between brightness and polarization vision suggest that P. xuthus perceive polarization angle as brightness, such that vertical polarization appears brighter than horizontal polarization.

Opsin expression, physiological characterization and identification of photoreceptor cells in the dorsal rim area and main retina of the desert locust, Schistocerca gregaria.

For compass orientation many insects rely on the pattern of sky polarization, but some species also exploit the sky chromatic contrast. Desert locusts, Schistocerca gregaria, detect polarized light through a specialized dorsal rim area (DRA) in their compound eye. To better understand retinal mechanisms underlying visual navigation, we compared opsin expression, spectral and polarization sensitivities and response-stimulus intensity functions in the DRA and main retina of the locust. In addition to previously characterized opsins of long-wavelength-absorbing (Lo1) and blue-absorbing visual pigments (Lo2), we identified an opsin of an ultraviolet-absorbing visual pigment (LoUV). DRA photoreceptors exclusively expressed Lo2, had peak spectral sensitivities at 441 nm and showed high polarization sensitivity (PS 1.3-31.7). In contrast, ommatidia in the main eye co-expressed Lo1 and Lo2 in five photoreceptors, expressed Lo1 in two proximal photoreceptors, and Lo2 or LoUV in one distal photoreceptor. Correspondingly, we found broadband blue- and green-peaking spectral sensitivities in the main eye and one narrowly tuned UV peaking receptor. Polarization sensitivity in the main retina was low (PS 1.3-3.8). V-log I functions in the DRA were steeper than in the main retina, supporting a role in polarization vision. Desert locusts occur as two morphs, a day-active gregarious and a night-active solitarious form. In solitarious locusts, sensitivities in the main retina were generally shifted to longer wavelengths, particularly in ventral eye regions, supporting a nocturnal lifestyle at low light levels. The data support the role of the DRA in polarization vision and suggest trichromatic colour vision in the desert locust.

Morphology and spectral sensitivity of long visual fibers and lamina monopolar cells in the butterfly Papilio xuthus.

Extensive analysis of the flower-visiting behavior of a butterfly, Papilio xuthus, has indicated complex interaction between chromatic, achromatic, and motion cues. Their eyes are spectrally rich with six classes of photoreceptors, respectively sensitive in the ultraviolet, violet, blue, green, red, and broad-band wavelength regions. Here, we studied the anatomy and physiology of photoreceptors and second-order neurons of P. xuthus, focusing on their spectral sensitivities and projection terminals to address where the early visual integration takes place. We thus found the ultraviolet, violet, and blue photoreceptors and all second-order neurons terminate in the distal region of the second optic ganglion, the medulla. We identified five types of second-order neurons based on the arborization in the first optic ganglion, the lamina, and the shape of the medulla terminals. Their spectral sensitivity is independent of the morphological types but reflects the combination of pre-synaptic photoreceptors. The results indicate that the distal medulla is the most plausible region for early visual integration.

Neurons innervating the lamina in the butterfly, Papilio xuthus.

The butterfly Papilio xuthus has compound eyes with three types of ommatidia. Each type houses nine spectrally heterogeneous photoreceptors (R1-R9) that are divided into six spectral classes: ultraviolet, violet, blue, green, red, and broad-band. Analysis of color discrimination has shown that P. xuthus uses the ultraviolet, blue, green, and red receptors for foraging. The ultraviolet and blue receptors are long visual fibers terminating in the medulla, whereas the green and red receptors are short visual fibers terminating in the lamina. This suggests that processing of wavelength information begins in the lamina in P. xuthus, unlike in flies. To establish the anatomical basis of color discrimination mechanisms, we examined neurons innervating the lamina by injecting neurobiotin into this neuropil. We found that in addition to photoreceptors and lamina monopolar cells, three distinct groups of cells project fibers into the lamina. Their cell bodies are located (1) at the anterior rim of the medulla, (2) between the proximal surface of the medulla and lobula plate, and (3) in the medulla cell body rind. Neurobiotin injection also labeled distinct terminals in medulla layers 1, 2, 3, 4 and 5. Terminals in layer 4 belong to the long visual fibers (R1, 2 and 9), while arbors in layers 1, 2 and 3 probably correspond to terminals of three subtypes of lamina monopolar cells, respectively. Immunocytochemistry coupled with neurobiotin injection revealed their transmitter candidates; neurons in (1) and a subset of neurons in (2) are immunoreactive to anti-serotonin and anti-γ-aminobutyric acid, respectively.

Rough eyes of the Northeast-Asian wood white, Leptidea amurensis.

The northeast-Asian wood white, Leptidea amurensis (Lepidoptera, Pieridae), belongs to the Dismorphiinae, a subfamily of the family Pieridae. We studied the structure of the compound eye in this species through a combination of anatomy, molecular biology and intracellular electrophysiology, with a particular focus on the evolution of butterfly eyes. We found that their eyes consist of three types of ommatidia, with a basic set of one short-, one middle- and one long-wavelength-absorbing visual pigment. The spectral sensitivities of the photoreceptors are rather simple, and peak in the ultraviolet, blue and green wavelength regions. The ommatidia have neither perirhabdomal nor fluorescent pigments, which modulate photoreceptor spectral sensitivities in a number of other butterfly species. These features are primitive, but the eyes of Leptidea exhibit another unique feature: the rough appearance of the ventral two-thirds of the eye. The roughness is due to the irregular distribution of facets of two distinct sizes. As this phenomenon exists only in males, it may represent a newly evolved sex-related feature.

Sex-specific retinal pigmentation results in sexually dimorphic long-wavelength-sensitive photoreceptors in the eastern pale clouded yellow butterfly, Colias erate.

The compound eyes of the eastern pale clouded yellow butterfly, Colias erate, contain three types of ommatidia (I, II and III), identifiable by the differing arrangements of pigment clusters around the rhabdoms. The pigment color is red in all ommatidial types except for type II ommatidia of females, where the pigment is orange. Intracellular recordings demonstrated that the spectral sensitivities of the proximal photoreceptors (R5-8) of all ommatidia in both sexes are strongly tuned by the perirhabdomal pigments. These pigments act as long-pass filters, shifting the peak sensitivities into the wavelength range above 600 nm. Due to the sex-specific pigments in type II ommatidia, the spectral sensitivities of the R5-8 photoreceptors of females peaked at 620 nm while those in males peaked at 660 nm. The measured spectral sensitivities could be well reproduced by an optical model assuming a long-wavelength-absorbing visual pigment with peak absorbance at 565 nm. Whereas the sexual dimorphism was unequivocally demonstrated for the ventral eye region, dimorphism in the dorsal region was not found. Presumably the ventral region is adapted for sexual behaviors such as courtship and oviposition.

'Color' processing in the butterfly visual system.

The swallowtail butterfly, Papilio xuthus, has excellent color discrimination abilities, and its visible light spectrum is notably wide. We discuss the neural basis of color vision in P. xuthus, highlighting some of the evolutionary adaptations in this species in relation to other insects. These adaptations include inter-photoreceptor (PR) interactions that produce spectral-opponent PRs, and complex higher order color-coding neurons.

Comparative morphology of serotonin-immunoreactive neurons innervating the central complex in the brain of dicondylian insects.

Serotonin (5-hydroxytryptamine) acts as a widespread neuromodulator in the nervous system of vertebrates and invertebrates. In insects, it promotes feeding, enhances olfactory sensitivity, modulates aggressive behavior, and, in the central complex of Drosophila, serves a role in sleep homeostasis. In addition to a role in sleep-wake regulation, the central complex has a prominent role in spatial orientation, goal-directed locomotion, and navigation vector memory. To further understand the role of serotonergic signaling in this brain area, we analyzed the distribution and identity of serotonin-immunoreactive neurons across a wide range of insect species. While one bilateral pair of tangential neurons innervating the central body was present in all species studied, a second type was labeled in all neopterans but not in dragonflies and firebrats. Both cell types show conserved major fiber trajectories but taxon-specific differences in dendritic targets outside the central body and axonal terminals in the central body, noduli, and lateral accessory lobes. In addition, numerous tangential neurons of the protocerebral bridge were labeled in all studied polyneopteran species except for Phasmatodea, but not in Holometabola. Lepidoptera and Diptera showed additional labeling of two bilateral pairs of neurons of a third type. The presence of serotonin in systems of columnar neurons apparently evolved independently in dragonflies and desert locusts. The data suggest distinct evolutionary changes in the composition of serotonin-immunolabeled neurons of the central complex and provides a promising basis for a phylogenetic study in a wider range of arthropod species.