Blue and double-peaked green receptors depend on ommatidial type in the eye of the Japanese yellow swallowtail Papilio xuthus

The compound eye of the butterfly Papilio xuthus is composed of three spectrally distinct types of ommatidia. We investigated the blue and double-peaked green receptors that are encountered distally in type I and III ommatidia, by means of intracellular recordings, in vivo fluorescence microscopy, and histology. The blue receptors are R1 and/or R2 photoreceptors; they contain the same mRNA encoding the opsin of the blue-absorbing visual pigment. However, here we found that the sensitivity in the UV wavelength region strongly depends on the ommatidial type; the blue receptors in type I ommatidia have a distinctly depressed UV sensitivity, which is attributed to lateral filtering in the fused rhabdom. In the main, fronto-ventral part of the eye, the R3 and R4 photoreceptors of all ommatidia contain the same set of two mRNAs encoding the opsins of green-absorbing visual pigments, PxL1 and PxL2. The spectral sensitivities are double-peaked, but the UV sensitivity of the R3 and R4 photoreceptors in type I ommatidia appears to be reduced, similar to that of the co-localized blue receptors.     

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.     

Photoreceptor Spectral Sensitivity in the Bumblebee,Bombus impatiens (Hymenoptera: Apidae)

The bumblebee Bombus impatiens is increasingly used as a model in comparative studies of colour vision, or in behavioural studies relying on perceptual discrimination of colour. However, full spectral sensitivity data on the photoreceptor inputs underlying colour vision are not available for B. impatiens. Since most known bee species are trichromatic, with photoreceptor spectral sensitivity peaks in the UV, blue and green regions of the spectrum, data from a related species, where spectral sensitivity measurements have been made, are often applied to B impatiens. Nevertheless, species differences in spectral tuning of equivalent photoreceptor classes may result in peaks that differ by several nm, which may have small but significant effects on colour discrimination ability. We therefore used intracellular recording to measure photoreceptor spectral sensitivity in B. impatiens. Spectral peaks were estimated at 347, 424 and 539 nm for UV, blue and green receptors, respectively, suggesting that this species is a UV-blue-green trichromat. Photoreceptor spectral sensitivity peaks are similar to previous measurements from Bombus terrestris, although there is a significant difference in the peak sensitivity of the blue receptor, which is shifted in the short wave direction by 12–13 nm in B. impatiens compared to B. terrestris.     

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.     

Polarization-based brightness discrimination in the foraging butterfly, Papilio xuthus

The human eye is insensitive to the angular direction of the light e-vector, but several animal species have the ability to discriminate differently polarized lights. How the polarization is detected is often unclear, however. Egg-laying Papilio butterflies have been shown to see false colours when presented with differently polarized lights. Here we asked whether this also holds in foraging butterflies. After training individuals to feed on nectar in front of an unpolarized spectral light, we carried out three dual-choice tests, where the discrimination of (i) the spectral content, (ii) the light intensity, and (iii) the e-vector orientation were investigated. In the first test, the butterflies selected the trained spectrum irrespective of its intensity, and in the second test they chose the light with the higher intensity. The result of the e-vector discrimination test was very similar to that of the second test, suggesting that foraging butterflies discriminate differently polarized lights as differing in brightness rather than as differing in colour. Papilio butterflies are clearly able to use at least two modes of polarization vision depending on the behavioural context.     

Behaviour and pleiotropy: Generalization of gene effects in the colour preferences of Japanese quail chicks (C. coturnix japonica)

Two lines of Japanese quail (Coturnix coturnix japonica) were artificially selected for colour preferences in 16 generations; one line for preference of blue over red, another for preference of red over blue. A genetic control line was maintained without selection, in parallel generations. Subjects of the blue-selected line exhibited general preferences for the shorter over the longer wavelengths, when tested with stimulus pairs of large hue differences. In similar situations red-line subjects preferred the shorter wavelength, within pairs above 542 nm. The relative strength of colour preferences in the three lines remained significantly different under conditions of both large and small hue differences. Genetic influences in preferences and preference, generalizations were detectable equally well with stimuli administered through wide-band gelatin filters and stimuli administered through narrow-band interference filters. In addition to demonstrating behavioural pleiotropy, the data counterindicated genetic variation in colour vision as a factor in the mediation of gene effects in the quail's colour preferences.     

Modellrechnung zur Datenverarbeitung beim Farbensehen des Menschen

Fig. 4 demonstrates the problem: How are the messages of the cones processed by the structures of the CNS before they are perceived in the form of colour impressions? The absorption curves of Fig. 2 are used as input functions of the three types of cones; they were adapted from the work of MacNichol et al. [2] and from similar results of other authors [1, 3, 4]. According to a well known hypothesis [8] the messages of the green receptors are supposed to be the antagonists to the messages of the “red” receptors, and the messages of the blue receptors the antagonists to the sum of the messages of the green and the “red” receptors (Fig. 11). But this hypothesis does not account for the red sensations present in the region of both the long and the short wave end of the visual spectrum (Fig. 14). Therefore another model was studied (Fig. 12): Blue (P 445, cyanolabe) being antagonistic to yellow (P 570, erythrolabe); and green (P 535, chlorolabe) being antagonistic to the combined action of blue and yellow (P 445 and P 570). This formalism explains the following experimental data: The distribution of colour sensations along the spectrum (Fig. 7, 13, 14); the wavelengths of antagonistic spectral colours (Fig. 5); the general form of the spectral hue discrimination function (Fig. 8); the number and the qualitative symptoms of colour deficiencies (Fig. 10, 18–24). A meaningful interpretation is given concerning the reduced colour vision of the distal parts of the retina.     

Out of the blue: the spectral sensitivity of hummingbird hawkmoths

The European hummingbird hawkmoth Macroglossum stellatarum is a diurnal nectar forager like the honeybee, and we expect similarities in their sensory ecology. Using behavioural tests and electroretinograms (ERGs), we studied the spectral sensitivity of M. stellatarum. By measuring ERGs in the dark-adapted eye and after adaptation to green light, we determined that M. stellatarum has ultraviolet (UV), blue and green receptors maximally sensitive at 349, 440 and 521 nm, and confirmed that green receptors are most frequent in the retina. To determine the behavioural spectral sensitivity (action spectrum) of foraging moths, we trained animals to associate a disk illuminated with spectral light, with a food reward, and a dark disk with no reward. While the spectral positions of sensitivity maxima found in behavioural tests agree with model predictions based on the ERG data, the sensitivity to blue light was 30 times higher than expected. This is different from the honeybee but similar to earlier findings in the crepuscular hawkmoth Manduca sexta. It may indicate that the action spectrum of foraging hawkmoths does not represent their general sensory capacity. We suggest that the elevated sensitivity to blue light is related to the innate preference of hawkmoths for blue flowers.     

How and why are uniformly polarization-sensitive retinae subject to polarization-related artefacts? Correction of some errors in the theory of polarization-induced false colours

If the photoreceptors of a colour vision system are polarization sensitive, the system detects polarization-induced false colours. Based on the functional similarities between polarization vision and colour vision, earlier it was believed that a uniformly polarization-sensitive (insect) retina (UPSR)-in which receptors of all spectral types have the same polarization sensitivity ratio and microvilli direction-cannot detect polarization-induced false colours. Here we show that, contrary to this belief, a colour vision based on a UPSR is subject to polarization-related artefacts, because both the degree and the angle of polarization of light reflected from natural surfaces depend on wavelength. Our second goal is to correct certain errors in the theory of polarizational false colours. The quantitative estimation of the influence of polarization sensitivity on colour vision was recently motivated by the suggestion that certain Papilio butterflies detect such false colours. The theoretical basis of this subject is to calculate the colour loci in the colour space of a visual system from the quantum catches of polarization-sensitive receptors of different spectral types. Horváth et al. (J. Exp. Biol. 205 (2002) 3281) gave the first exact mathematical and receptor-physiological derivation of formulae for these calculations. Here we prove that the two formulae given earlier by others are inappropriate or erroneous. This, however, does not influence the validity of the experimental data and the principal conclusions drawn about the colour vision and polarization sensitivity in Papilio butterflies.     

The photoreceptor localization confirms the spectral heterogeneity of ommatidia in the male small white butterfly,<Emphasis Type="Italic"> Pieris rapae crucivora</Emphasis>

The compound eye of Pieris rapae crucivora contains ventrally three types of histologically distinct ommatidia. An ommatidium contains nine photoreceptors, four of which (R1-4) construct the distal tier of the rhabdom. We determined the sensitivity spectra of the R1-4 distal photoreceptors in each type of ommatidia by intracellular electrophysiology and identified UV, blue, double-peaked blue, green, and a green receptor with depressed sensitivity in the violet. We localized these receptors in each type of ommatidia by injecting dye after the recording. In type I ommatidia the R1 and R2 cells are UV and blue receptors. When R1 is UV sensitive, R2 is always blue sensitive, or vice versa. R3 and R4 in type I are both green receptors. In type II, R1 and R2 are both double-peaked blue receptors and R3 and R4 are both green receptors with depressed sensitivity in the violet. In type III, R1 and R2 are both UV, and R3 and R4 are green receptors. The double-peaked blue, and green receptors with depressed sensitivity in the violet in type II ommatidia have depressed sensitivity at 420 nm, which is probably due to the filtering effect of a fluorescing material present in the type II ommatidia. Spectral heterogeneity of ommatidia seems to be a common design of insect compound eyes.     

Photoreceptor processing speed and input resistance changes during light adaptation correlate with spectral class in the bumblebee, Bombus impatiens

Colour vision depends on comparison of signals from photoreceptors with different spectral sensitivities. However, response properties of photoreceptor cells may differ in ways other than spectral tuning. In insects, for example, broadband photoreceptors, with a major sensitivity peak in the green region of the spectrum (>500 nm), drive fast visual processes, which are largely blind to chromatic signals from more narrowly-tuned photoreceptors with peak sensitivities in the blue and UV regions of the spectrum. In addition, electrophysiological properties of the photoreceptor membrane may result in differences in response dynamics of photoreceptors of similar spectral class between species, and different spectral classes within a species. We used intracellular electrophysiological techniques to investigate response dynamics of the three spectral classes of photoreceptor underlying trichromatic colour vision in the bumblebee, Bombus impatiens, and we compare these with previously published data from a related species, Bombus terrestris. In both species, we found significantly faster responses in green, compared with blue- or UV-sensitive photoreceptors, although all 3 photoreceptor types are slower in B. impatiens than in B. terrestris. Integration times for light-adapted B. impatiens photoreceptors (estimated from impulse response half-width) were 11.3 ± 1.6 ms for green photoreceptors compared with 18.6 ± 4.4 ms and 15.6 ± 4.4 for blue and UV, respectively. We also measured photoreceptor input resistance in dark- and light-adapted conditions. All photoreceptors showed a decrease in input resistance during light adaptation, but this decrease was considerably larger (declining to about 22% of the dark value) in green photoreceptors, compared to blue and UV (41% and 49%, respectively). Our results suggest that the conductances associated with light adaptation are largest in green photoreceptors, contributing to their greater temporal processing speed. We suggest that the faster temporal processing of green photoreceptors is related to their role in driving fast achromatic visual processes.     

Blue–green opponency and trichromatic vision in the greenhouse whitefly (Trialeurodes vaporariorum) explored using light emitting diodes

Visual orientation in the greenhouse whitefly (Trialeurodes vaporariorum Westwood, Hemiptera: Aleyrodidae) is the result of “wavelength‐specific behaviours.” Green–yellow elicits “settling behaviour” while ultraviolet (UV) radiation initiates “migratory behaviour.” The only available physiological study of the photoreceptors' spectral efficiency showed peaks in the green and the UV range and whitefly vision was said to be dichromatic so far. In order to study the visual behaviour of T. vaporariorum, 19 narrow‐bandwidth light emitting diodes (LEDs) covering the UV‐A and visible range were used in combination with light scattering acrylic glass screens in a small‐scale choice arena under greenhouse conditions. Multiple‐choice and dual‐choice assays were performed, resulting in LED‐based behavioural action spectra of settling (green) and migratory behaviour (UV). A potential inhibitory blue–green chromatic mechanism was studied by combining yellow with different bluish LEDs. Intensity dependencies were illustrated by changing LED intensities. Regarding the “settling response,” highest attraction was achieved by a green LED with a centroid wavelength of 550 nm, while a blue LED with 469 nm proved to be most inhibitory. Besides this inhibitory interaction, an intensity dependence was observed within the action spectrum in the green–yellow range. “Migratory behaviour” was elicited the most by the UV LED with the shortest available wavelength of 373 nm. The results provide compelling behavioural evidence for the presence of a green and a yet undescribed blue sensitive photoreceptor and a blue–green opponent mechanism. Furthermore, empirical colour choice models were built and receptor peaks were estimated around 510–520 nm (green), 480–490 nm (blue) and 340–370 nm (UV). Consequently, a trichromatic receptor setup is suggested for T. vaporariorum.     

Colour discrimination in dim light by the larvae of the African catfish <Emphasis Type="Italic">Clarias gariepinus</Emphasis>

Many demersal fish species undergo vertical shifts in habitats during ontogeny especially after larval metamorphosis. The visual spectral sensitivity shifts with the habitat, indicating a change in colour vision. Colour vision depends on sufficient ambient light and becomes ineffective at a particular low light intensity. It is not known how fishes see colour in dim light. By means of a behavioural experiment on larval African catfish Clarias gariepinus in the laboratory, we determined colour vision and colour discrimination in dim light. Light-adapted larvae were subjected to classical conditioning to associate a reward feed with a green or a red stimulus placed among 7 shades of grey. The larvae learned this visual task after 70 and 90 trials. A different batch of larvae were trained to discriminate between green and red and then tested for the ability to discriminate between these colours, as the light intensity was reduced. The larvae learned this visual task after 110 trials in bright light and were able to discriminate colours, as light was dimmed until 0.01 lx, the minimal illuminance measurable in this study, and similar to starlight. The retinae of the larvae were found to be light adapted at 0.01 lx; thus indicating cone-based colour vision at this illuminance. For comparison, three human subjects were tested under similar conditions and showed a colour vision threshold at between 1.5 and 0.1 lx. For the larvae of C. gariepinus, the ability of colour discrimination in dim light is probably due to its retinal tapetum, which could increase the sensitivity of cones.     

Blue colour preference in honeybees distracts visual attention for learning closed shapes

Spatial vision is an important cue for how honeybees (Apis mellifera) find flowers, and previous work has suggested that spatial learning in free-flying bees is exclusively mediated by achromatic input to the green photoreceptor channel. However, some data suggested that bees may be able to use alternative channels for shape processing, and recent work shows conditioning type and training length can significantly influence bee learning and cue use. We thus tested the honeybees’ ability to discriminate between two closed shapes considering either absolute or differential conditioning, and using eight stimuli differing in their spectral characteristics. Consistent with previous work, green contrast enabled reliable shape learning for both types of conditioning, but surprisingly, we found that bees trained with appetitive-aversive differential conditioning could additionally use colour and/or UV contrast to enable shape discrimination. Interestingly, we found that a high blue contrast initially interferes with bee shape learning, probably due to the bees innate preference for blue colours, but with increasing experience bees can learn a variety of spectral and/or colour cues to facilitate spatial learning. Thus, the relationship between bee pollinators and the spatial and spectral cues that they use to find rewarding flowers appears to be a more rich visual environment than previously thought.     

Colour perception of single and mixed monochromatic lights in the blowfly Lucilia cuprina

Colour perception of spectral lights and mixtures of two monochromatic lights of blue and yellow wavelengths was studied in the blowfly Lucilia cuprina by using a generalization test in which the fly had to compare these lights in memory with coloured papers (blue, green, yellow and red) represented in the test array. Flies trained to a monochromatic light in the wavelength range of 429–491 nm responded to blue; those trained to 502–511 nm to green; and those trained to 522–582 nm to yellow. The maximal generalization for blue was found at 429 nm and that for yellow at 543 nm. Flies trained to the mixtures responded neither to blue, green nor yellow, when the blue component was mixed with the yellow component in a ratio of approximately 1 3. It seems that the fly perceives the mixtures as a neutral or an achromatic light. Colour loci of coloured papers, spectral lights and mixtures of two monochromatic lights used formed blue, yellow and neutral clusters in a colour triangle with respect to generalization responses to test colours.     

How Bees Discriminate a Pattern of Two Colours from Its Mirror Image

A century ago, in his study of colour vision in the honeybee (Apis mellifera), Karl von Frisch showed that bees distinguish between a disc that is half yellow, half blue, and a mirror image of the same. Although his inference of colour vision in this example has been accepted, some discrepancies have prompted a new investigation of the detection of polarity in coloured patterns. In new experiments, bees restricted to their blue and green receptors by exclusion of ultraviolet could learn patterns of this type if they displayed a difference in green contrast between the two colours. Patterns with no green contrast required an additional vertical black line as a landmark. Tests of the trained bees revealed that they had learned two inputs; a measure and the retinotopic position of blue with large field tonic detectors, and the measure and position of a vertical edge or line with small-field phasic green detectors. The angle between these two was measured. This simple combination was detected wherever it occurred in many patterns, fitting the definition of an algorithm, which is defined as a method of processing data. As long as they excited blue receptors, colours could be any colour to human eyes, even white. The blue area cue could be separated from the green receptor modulation by as much as 50°. When some blue content was not available, the bees learned two measures of the modulation of the green receptors at widely separated vertical edges, and the angle between them. There was no evidence that the bees reconstructed the lay-out of the pattern or detected a tonic input to the green receptors.     

Learning and discrimination of coloured papers in the walking blowfly,Lucilia cuprina

Summary A new training and testing paradigm for walking sheep blowflies, Lucilia cuprina, is described. A fly is trained by presenting it with a droplet of sugar solution on a patch of coloured paper. After having consumed the sugar droplet, the fly starts a systematic search. While searching, it is confronted with an array of colour marks consisting of four colours displayed on the test cardboard (Fig. 1). Colours used for training and test include blue, green, yellow, orange, red, white and black.Before training, naive flies are tested for their spontaneous colour preferences on the test array. Yellow is visited most frequently, green least frequently (Table 2). Spontaneous colour preferences do not simply depend on subjective brightness (Table 1).The flies trained to one of the colours prefer this colour significantly (Figs. 5 and 9–11). This behaviour reflects true learning rather than sensitisation (Figs. 6–7). The blue and yellow marks are learned easily and discriminated well (Figs. 5, 9, 11). White is also discriminated well, although the response frequencies are lower than to blue and yellow (Fig. 11). Green is discriminated from blue but weakly from yellow and orange (Figs. 5, 9, 10). Red is a stimulus as weak as black (Figs. 8, 9). These features of colour discrimination reflect the spectral loci of colours in the colour triangle (Fig. 14).The coloured papers seem to be discriminated mainly by the hue of colours (Fig. 12), but brightness may also be used to discriminate colour stimuli (Fig. 13).     

Bat Eyes Have Ultraviolet-Sensitive Cone Photoreceptors

Mammalian retinae have rod photoreceptors for night vision and cone photoreceptors for daylight and colour vision. For colour discrimination, most mammals possess two cone populations with two visual pigments (opsins) that have absorption maxima at short wavelengths (blue or ultraviolet light) and long wavelengths (green or red light). Microchiropteran bats, which use echolocation to navigate and forage in complete darkness, have long been considered to have pure rod retinae. Here we use opsin immunohistochemistry to show that two phyllostomid microbats, Glossophaga soricina and Carollia perspicillata, possess a significant population of cones and express two cone opsins, a shortwave-sensitive (S) opsin and a longwave-sensitive (L) opsin. A substantial population of cones expresses S opsin exclusively, whereas the other cones mostly coexpress L and S opsin. S opsin gene analysis suggests ultraviolet (UV, wavelengths <400 nm) sensitivity, and corneal electroretinogram recordings reveal an elevated sensitivity to UV light which is mediated by an S cone visual pigment. Therefore bats have retained the ancestral UV tuning of the S cone pigment. We conclude that bats have the prerequisite for daylight vision, dichromatic colour vision, and UV vision. For bats, the UV-sensitive cones may be advantageous for visual orientation at twilight, predator avoidance, and detection of UV-reflecting flowers for those that feed on nectar.     

Spectral and polarized light sensitivity of photoreceptors in the compound eye of the cricket (Gryllus bimaculatus)

Summary Retinula cells in the compound eye of the cricket (Gryllus bimaculatus) were recorded intracellularly and stained with Lucifer yellow. Two different methods were used to determine the spectral sensitivity of these cells: a) the spectral scanning method, and b) the conventional flash method. Three spectral types, with S()-curves close to the rhodopsin-absorption functions, were found with _max at 332 nm (UV), 445 nm (blue) and 515 nm (green), respectively.Blue receptors were only recorded in the anatomically specialized dorsal rim area (DRA), and UV and green receptors in the dorsal region of the pigmented part of the eye, whereby green receptors were only found in the ventral eye. On the basis of these results, model calculations are presented for di- and trichromatic colour vision in the cricket.The fluorescence markings revealed green receptors whose axons project with short visual fibres to the lamina, and a UV receptor with a long visual fibre which projects through the lamina to the medulla. The blue receptors send their axons either to the lamina and medulla (long visual fibres) or only to the lamina (short visual fibres).The temporal dynamics of the three receptor types were examined. The blue receptors lack a phasic component of the receptor potential, and the time from stimulus on-set to peak potential is strongly increased compared to the UV and green receptors. Light adaptation reduces the latency to less than half of the dark adapted state.Spectral adaptation experiments revealed an unidirectional coupling between UV and green receptors, and it was found that polarization sensitivity (PS) in blue cells was much higher (PS= 6.5±1.5) than that of UV (PS=1.76±0.05) and green (2.26±0.57) receptors. The functional aspects of the three receptor types are discussed with respect to the presented physiological and morphological data.Abbreviations DA dorsal area - DRA dorsal rim area - PS polarization sensitivity     

Spectral sensitivities including the ultraviolet of the passeriform bird Leiothrix lutea

Spectral sensitivity functions of a passeriform bird, the Red-billed Leiothrix Leiothrix lutea (Timalidae) were determined in a behavioural test under different background illuminations.