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.     

Simultaneous brightness contrast of foraging Papilio butterflies

This study focuses on the sense of brightness in the foraging Japanese yellow swallowtail butterfly, Papilio xuthus. We presented two red discs of different intensity on a grey background to butterflies, and trained them to select one of the discs. They were successfully trained to select either a high intensity or a low intensity disc. The trained butterflies were tested on their ability to perceive brightness in two different protocols: (i) two orange discs of different intensity presented on the same intensity grey background and (ii) two orange discs of the same intensity separately presented on a grey background that was either higher or lower in intensity than the training background. The butterflies trained to high intensity red selected the orange disc of high intensity in protocol 1, and the disc on the background of low intensity grey in protocol 2. We obtained similar results in another set of experiments with purple discs instead of orange discs. The choices of the butterflies trained to low intensity red were opposite to those just described. Taken together, we conclude that Papilio has the ability to learn brightness and darkness of targets independent of colour, and that they have the so-called simultaneous brightness contrast.     

Tetrachromacy in a butterfly that has eight varieties of spectral receptors

This paper presents the first evidence of tetrachromacy among invertebrates. The Japanese yellow swallowtail butterfly, Papilio xuthus, uses colour vision when foraging. The retina of Papilio is furnished with eight varieties of spectral receptors of six classes that are the ultraviolet (UV), violet, blue (narrow-band and wide-band), green (single-peaked and double-peaked), red and broad-band classes. We investigated whether all of the spectral receptors are involved in colour vision by measuring the wavelength discrimination ability of foraging Papilio. We trained Papilio to take nectar while seeing a certain monochromatic light. We then let the trained Papilio choose between two lights of different wavelengths and determined the minimum discriminable wavelength difference Deltalambda. The Deltalambda function of Papilio has three minima at approximately 430, 480 and 560nm, where the Deltalambda values approximately 1nm. This is the smallest value found for wavelength discrimination so far, including that of humans. The profile of the Deltalambda function of Papilio can be best reproduced by postulating that the UV, blue (narrow-band and wide-band), green (double-peaked) and red classes are involved in foraging. Papilio colour vision is therefore tetrachromatic.     

Rhabdom evolution in butterflies: insights from the uniquely tiered and heterogeneous ommatidia of the Glacial Apollo butterfly, Parnassius glacialis

The eye of the Glacial Apollo butterfly, Parnassius glacialis, a 'living fossil' species of the family Papilionidae, contains three types of spectrally heterogeneous ommatidia. Electron microscopy reveals that the Apollo rhabdom is tiered. The distal tier is composed exclusively of photoreceptors expressing opsins of ultraviolet or blue-absorbing visual pigments, and the proximal tier consists of photoreceptors expressing opsins of green or red-absorbing visual pigments. This organization is unique because the distal tier of other known butterflies contains two green-sensitive photoreceptors, which probably function in improving spatial and/or motion vision. Interspecific comparison suggests that the Apollo rhabdom retains an ancestral tiered pattern with some modification to enhance its colour vision towards the long-wavelength region of the spectrum.     

Action spectrum of foraging behavior of the Japanese yellow swallowtail butterfly, Papilio xuthus

This paper describes the action spectrum of foraging behavior of a butterfly, Papilio xuthus. We first established an experimental protocol to evaluate learning and discrimination of monochromatic light by the butterflies. We trained butterflies to feed on sucrose solution at the window illuminated with certain monochromatic light produced through a monochromator. After confirming that they learned the monochromatic light, after 10 days of training, we tested the butterflies one by one. We presented training wavelengths for each individual at different intensities, and recorded whether they perform foraging behavior under freely-flying as well as tethered conditions. Freely-flying butterflies responded to light by visiting the window and searching for nectar around it, whereas tethered butterflies responded by extending their proboscides towards the window. The light intensity required to elicit 50% response for each tested monochromatic light was plotted. The resulting action spectrum for the visit was rather flat with the maximum sensitivity a 420 nm, whereas the spectrum for the proboscis extension had prominent peaks at 380, 500 and 600 nm. The difference in action spectra indicates that the visit and the proboscis extension are controlled by two independent mechanisms at least in part.     

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.     

Spectral organization of the eye of a butterfly,Papilio

Journal of Comparative Physiology A - This review outlines our recent studies on the spectral organization of butterfly compound eyes, with emphasis on the Japanese yellow swallowtail butterfly,...     

Colour cues proved to be more informative for dogs than brightness

The results of early studies on colour vision in dogs led to the conclusion that chromatic cues are unimportant for dogs during their normal activities. Nevertheless, the canine retina possesses two cone types which provide at least the potential for colour vision. Recently, experiments controlling for the brightness information in visual stimuli demonstrated that dogs have the ability to perform chromatic discrimination. Here, we show that for eight previously untrained dogs colour proved to be more informative than brightness when choosing between visual stimuli differing both in brightness and chromaticity. Although brightness could have been used by the dogs in our experiments (unlike previous studies), it was not. Our results demonstrate that under natural photopic lighting conditions colour information may be predominant even for animals that possess only two spectral types of cone photoreceptors.     

Flower constancy and learning in foraging preferences of the green-veined white butterfly Pleris napi

Abstract.

• 1 Evolutionary pressure should select for efficient foraging strategies, within the constraints of other selective forces. We assess the mechanisms underlying flower choice in the butterfly, Pieris napi (L.), which as an adult forages for nectar. Experiments were carried out on a laboratory colony, using artificial flowers of two colours, and replicated on two successive generations.
• 2 When nectar was freely available from all flowers, equal numbers of butterflies visited each colour, but individual butterflies exhibited flower constancy, showing a strong preference for one colour or the other.
• 3 Following 3 day conditioning periods in which nectar was available from flowers of one colour only, butterflies responded by developing a preference for this colour, which persisted when both flower colours were refilled. This preference could subsequently be switched to the other flower colour following a further 3 days of conditioning. These are interpreted as adaptive (learned) responses, which would have obvious selective benefits in the field, enabling butterflies to avoid flower species which experience has shown are poor sources of nectar, and to adapt to temporal and spatial changes in nectar availability.

     

A novel retinol-binding protein in the retina of the swallowtail butterfly, Papilio xuthus.

Retinoid-binding proteins are indispensable for visual cycles in both vertebrate and invertebrate retinas. These proteins stabilize and transport hydrophobic retinoids in the hydrophilic environment of plasma and cytoplasm, and allow regeneration of visual pigments. Here, we identified a novel retinol-binding protein in the eye of a butterfly, Papilio xuthus. The protein that we term Papilio retinol-binding protein (Papilio RBP) is a major component of retinal soluble proteins and exclusively binds 3-hydroxyretinol, and emits fluorescence peaking at 480 nm under ultraviolet (UV) illumination. The primary structure, deduced from the nucleotide sequence of the cDNA, shows no similarity to any other lipophilic ligand-binding proteins. The molecular mass and isoelectric point of the protein estimated from the amino-acid sequence are 26.4 kDa and 4.92, respectively. The absence of any signal sequence for secretion in the N-terminus suggests that the protein exists in the cytoplasmic matrix. All-trans 3-hydroxyretinol is the major ligand of the Papilio RBP in dark-adapted eyes. Light illumination of the eyes increases the 11-cis isomer of the ligand and induces redistribution of the Papilio RBP from the proximal to the distal part of the photoreceptor layer. These results suggest that the Papilio RBP is involved in visual pigment turnover.     

Population dynamics of the swallowtail butterfly,Papilio xuthus L., in a deforested area

The long-term population dynamics of the swallowtail butterfly, Papilio xuthus, were studied by means of life table analysis in a deforested area, where the host tree, Zanthoxylum ailanthoides, was growing. The number of eggs laid on host trees in the deforested area decreases as secondary succession progresses. When the trees were classified into three groups, i. e. tall, medium and short, according to their height relative to the surrounding vegetation, less eggs were laid on tall than on short host trees. Life tables for the natural populations in each generation were developed on the basis of mean values for 6 years. Eggs and larvae in early stages were attacked chiefly by small- and middle-sized predators, such as ants, spiders, bugs and orthopterids, while later stage larvae were attacked chiefly by birds or Polistes wasps. An inchneumonid wasp was the most important mortality factor in the pupal stage. Key-factor analysis was tested on the life table data of each immature stage. The late larval stage was a key stage, and most mortality factors were considered to operate independently. The test for density dependency did not show any tendencies. The analysis of variance in two-way classifications was carried out for the difference of survival rate among the tree groups. It was suggested that tall host trees were unsuitable for P. xuthus. The construction of life tables for artificially inoculated populations on large host trees showed that high physiological death rate were characteristic of such trees in comparison with natural populations on smaller trees. The characteristics of the population dynamics of P. xuthus in the deforested area are compared among some kinds of the habitat, and the interrelationship between P. xuthus and its host plant are discussed.     

Properties of Orange-Pupa-Inducing Factor (OPIF) in the swallowtail butterfly, Papilio xuthus L

Diapause pupae of the swallowtail butterfly Papilio xuthus L. exhibit diapause-green, orange and brownish-orange color polymorphism. Development of orange pupae involves a neuroendocrine factor inducing orange pupa (Orange-Pupa-Inducing Factor, OPIF), which is secreted from the head-thoracic region during late pharate pupal stages, in particular from the ganglia of short-day animals located posteriorly from the second thoracic ganglion2 (TG2). This report describes certain properties of OPIF using bioassays involving ligated abdomens of short-day pharate pupae. Localization of OPIF in the central nervous system of short-day larvae indicated that it was present predominantly in TG2, thoracic ganglion3 (TG3) and abdominal ganglion1 (AG1) complexes. OPIF activity in TG(2,3)-AG1 complexes was over two times higher than in the more posteriorely located ganglia. The developmental profile of OPIF in last instar short-day larvae revealed that OPIF activity in larval ganglia posterior to TG2 became gradually higher as larval growth proceeded, suggesting that OPIF might be accumulated in TG(2,3)-AG(1-7) complexes as larvae prepare for pupal molting. Furthermore, ligated abdomens of short-day larvae developed into pupae of an orange type when a 2% NaCl extract containing OPIF prepared from TG(2,3)-AG(1-7) complexes of long-day larvae was injected into ligated abdomens of short-day pharate pupae, indicating that OPIF is also present in long-day larvae. Additionally, a biochemical investigation using gel filtration chromatography showed that the molecular weight of OPIF was about 10 kDa.     

Interactions of environmental factors influencing pupal coloration in swallowtail butterfly Papilio xuthus

The swallowtail butterfly Papilio xuthus Linné [Lepidoptera: Papilionidae] exhibits pupal protective color polyphenism. Interactions of various environmental factors on pupal coloration were analyzed in non-diapausing individuals. Under sufficient light (200lux), most pupating larvae became green pupae when the surface of the pupation site was smooth, while they became brown when the surface was rough. Tactile signals are the positive environmental factors causing induction of the brown pupal coloration. In dark boxes, the induction of the brown pupal coloration was easily induced even on a smooth surface, suggesting that light suppresses induction of brown coloration. Different colors of pupation sites did not affect pupal coloration under sufficient light. Environmental factors received during a critical period both before girdling and after girdling affected pupal coloration. When tactile signals received from rough surfaces reach threshold levels during pupation, brown pupal coloration is determined. Larvae reared under a daily periodicity of natural light formed a girdle at midnight, subsequently, the prepupae received strong daylight the following day. Under natural light most larvae produced brown pupae on rough surfaces and green pupae on smooth surfaces.     

Purification and Characterization of Biliverdin-binding Protein from Larval Hemolymph of the Swallowtail Butterfly,Papilio xuthus L.

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The N-amino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.     

Multiple matings increase the fecundity of the yellow swallowtail butterfly,Papilio xuthus L., in summer generations

Females of the yellow swallowtail butterfly, Papilio xuthus,were reared in the laboratory. They were divided into four groups held under different mating conditions: nonmating (virgin) and mated once, twice, and three times. The number of eggs in the ovaries was counted by dissection. Virgin females produced increasing numbers of mature eggs, up to about 30, in the week following emergence. When the female had mated once, the number of mature eggs was significantly higher than that of virgin females by the second day after emergence. However, the double- and triplemated females did not increase the number of eggs in each state further than the singlemated females. The double-mated females deposited significantly more eggs than the singlemated females in the laboratory. The triplemated females also deposited more eggs on the day after the third mating than the doublemated females. Thus, multiple matings increased the number of eggs deposited. The change in the hatchability and the morphology of the spermatophore in the bursa copulatrix suggested that the sperm from the last mating had precedence.     

Perturbation of the wing color pattern of a swallowtail butterfly,Papilio xuthus,induced by acid carboxypeptidase

Hypodermal injection of Toughmac-E, a digestive mixture composed of nine digestive components, or Molsin induced perturbation of the wing color pattern in 0- to 2-day-old pupae of Papilio xuthus, but had no effect on prepupae or 3- to 4-day-old pupae. The effective component in Toughmac-E was identified as Molsin, an acid carboxypeptidase of Aspergillus saitoi which specifically liberates tyrosine and phenylalanine from the C-terminal residues of proteins. The pattern perturbation occurred in either side of the fore- and hindwings of both sexes. When this enzyme was administered, stronger melanization than in the normal wings was found in the whole wings of most butterflies, but in other butterflies, the yellow region was enlarged. These findings suggest that the pattern perturbation was caused by changes in the levels of melanin and papiliochromes in scales. Melanin is a black pigment and papiliochromes are yellow pigments; their common precursor is dopamine. The normal pattern is formed by a predetermined balance of melanin and papiliochromes, whereas the deposit of an excess amount of tyrosine and/or phenylalanine disturbs this balance and results in perturbation of the color pattern. Administration of L-dopa or dopamine had no effect on the wing pattern. When the activity of an endogenous acid carboxypeptidase similar to Molsin appears in the early pupa, the summed activities of the endogenous and exogenous acid carboxypeptidases must induce a pattern perturbation. The relations between the endogenous acid carboxypeptidase and its probable substrate, the reserve protein, and the physiological roles of these relations in the regulation, utilization and excretion of amino acids are discussed.     

Purification and characterization of biliverdin-binding protein from larval hemolymph of the swallowtail butterfly, Papilio xuthus L

The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The Namino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.