Butterfly Papilio Xuthus Research Articles

Motion-sensitive neurons activated by chromatic contrast in a butterfly visual system.

A pattern of two equally bright colours contains only chromatic contrast. Unlike in flies, such a pattern elicits strong optokinetic responses in the butterfly Papilio xuthus. To investigate the neural basis of chromatic motion vision, we performed single-cell electrophysiology. We found spiking neurons exhibiting direction-selective motion sensitivity in the second optic ganglion, the medulla. We analysed the response characteristics of these neurons using two-colour stripe patterns moving vertically. We systematically manipulated the intensities of the colours so that the set of presented patterns included an isoluminant condition for the butterfly. Moving patterns containing only chromatic contrast still elicited a response in the neurons. The neurons' sensitivity profile is similar to that of the behavioural responses. Post-recording dye injection revealed that the neurons have dendrites in the ventral lateral protocerebrum and axonal processes in the medulla, suggesting a feedback role. Presumably, the neurons contribute to subtracting wide-field motion to facilitate the detection of small moving targets. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

Chromatic information processing in the first optic ganglion of the butterfly Papilio xuthus

The butterfly Papilio xuthus has acute tetrachromatic color vision. Its eyes are furnished with eight spectral classes of photoreceptors, situated in three types of ommatidia, randomly distributed in the retinal mosaic. Here, we investigated early chromatic information processing by recording spectral, angular, and polarization sensitivities of photoreceptors and lamina monopolar cells (LMCs). We identified three spectral classes of LMCs whose spectral sensitivities corresponded to weighted linear sums of the spectral sensitivities of the photoreceptors present in the three ommatidial types. In ~ 25% of the photoreceptor axons, the spectral sensitivities differed from those recorded at the photoreceptor cell bodies. These axons showed spectral opponency, most likely mediated by chloride ion currents through histaminergic interphotoreceptor synapses. The opponency was most prominent in the processes of the long visual fibers in the medulla. We recalculated the wavelength discrimination function using the noise-limited opponency model to reflect the new spectral sensitivity data and found that it matched well with the behaviorally determined function. Our results reveal opponency at the first stage of Papilio’s visual system, indicating that spectral information is preprocessed with signals from photoreceptors within each ommatidium in the lamina, before being conveyed downstream by the long visual fibers and the LMCs.

Immunolocalization suggests a role of the histamine-gated chloride channel PxHCLB in spectral opponent processing in butterfly photoreceptors.

Spectrally opponent responses, that is, wavelength-dependent inversions of response polarity, have been observed at the level of photoreceptors in butterflies. As inter-photoreceptor connections have been found in the butterfly Papilio xuthus, and histamine is the only neurotransmitter so far identified in insect photoreceptors, we hypothesize that histaminergic sign-inverting synapses exist in the lamina between different spectral receptors as a mechanism for spectral opponency as in the medulla of Drosophila. Here, we localized two histamine-gated chloride channels, PxHCLA (Drosophila Ort homolog) and PxHCLB (Drosophila HisCl1 homolog), in the visual system of Papilio xuthus by using specific antisera. The antiPxHCLA labeling was associated with the membrane of nonphotoreceptor cells that are postsynaptic to photoreceptors, while the antiPxHCLB labeling overlapped with photoreceptor axons, indicating that PxHCLB is expressed at inter-photoreceptor synapses: PxHCLB is likely involved in producing spectral opponency at the first visual synapses. Color processing in Papilio may appear earlier than previously hypothesized in insect visual systems.

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.

Physiological responses of ionotropic histamine receptors, PxHCLA and PxHCLB, to neurotransmitter candidates in a butterfly, Papilio xuthus.

Histamine is the only known neurotransmitter released by arthropod photoreceptors. Synaptic transmission from photoreceptors to second-order neurons is mediated by the activation of histamine-gated chloride channels (HCLs). These histaminergic synapses have been assumed to be conserved among insect visual systems. However, our understanding of the channels in question has thus far been based on studies in flies. In the butterfly Papilio xuthus, we have identified two candidate histamine-gated chloride channels, PxHCLA and PxHCLB, and studied their physiological properties using a whole-cell patch-clamp technique. We studied the responses of channels expressed in cultured cells to histamine as well as to other neurotransmitter candidates, namely GABA, tyramine, serotonin, d-/l-glutamate and glycine. We found that histamine and GABA activated both PxHCLA and PxHCLB, while the other molecules did not. The sensitivity to histamine and GABA was consistently higher in PxHCLB than in PxHCLA. Interestingly, simultaneous application of histamine and GABA activated both PxHCLA and PxHCLB more strongly than either neurotransmitter individually; histamine and GABA may have synergistic effects on PxHCLs in the regions where they co-localize. Our results suggest that the physiological properties of the histamine receptors are basically conserved among insects, but that the response to GABA differs between butterflies and flies, implying variation in early visual processing among species.

Unique Temporal Expression of Triplicated Long-Wavelength Opsins in Developing Butterfly Eyes.

Following gene duplication events, the expression patterns of the resulting gene copies can often diverge both spatially and temporally. Here we report on gene duplicates that are expressed in distinct but overlapping patterns, and which exhibit temporally divergent expression. Butterflies have sophisticated color vision and spectrally complex eyes, typically with three types of heterogeneous ommatidia. The eyes of the butterfly Papilio xuthus express two green- and one red-absorbing visual pigment, which came about via gene duplication events, in addition to one ultraviolet (UV)- and one blue-absorbing visual pigment. We localized mRNAs encoding opsins of these visual pigments in developing eye disks throughout the pupal stage. The mRNAs of the UV and blue opsin are expressed early in pupal development (pd), specifying the type of the ommatidium in which they appear. Red sensitive photoreceptors first express a green opsin mRNA, which is replaced later by the red opsin mRNA. Broadband photoreceptors (that coexpress the green and red opsins) first express the green opsin mRNA, later change to red opsin mRNA and finally re-express the green opsin mRNA in addition to the red mRNA. Such a unique temporal and spatial expression pattern of opsin mRNAs may reflect the evolution of visual pigments and provide clues toward understanding how the spectrally complex eyes of butterflies evolved.

Wavelength discrimination in the hummingbird hawkmoth Macroglossum stellatarum

Despite the strong relationship between insect vision and the spectral properties of flowers, the visual system has been studied in detail in only a few insect pollinator species. For instance, wavelength discrimination thresholds have been determined in two species only: the honeybee (Apis mellifera) and the butterfly Papilio xuthus. Here, we present the wavelength discrimination thresholds (Δλ) for the hawkmoth Macroglossum stellatarum. We compared the data with those found for the honeybee, the butterfly P. xuthus and the predictions of a colour discrimination model. After training moths to feed from a rewarded disc illuminated with a monochromatic light, we tested them in a dual-choice situation, in which they had to choose between light of the training wavelength and a novel unrewarded wavelength. To characterise the Δλ function, we decreased the difference between wavelengths in subsequent tests. We also varied the light intensity to test its effect on the discrimination capacity. In agreement with the predictions of the model, we found two expected minima of discrimination where photoreceptor sensitivities overlap, as well as a third, minor, unpredicted minimum around the peak of the blue photoreceptor. Macroglossum stellatarum is capable of discriminating between lights with a wavelength difference of 1-2 nm. These discrimination minima are similar to those found for the tetrachromatic P. xuthus, and are better than those of the honeybee. The moth is also capable of using achromatic information to discriminate between lights of long wavelengths.

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.

The mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) and related phylogenetic analyses

The nearly complete mitochondrial genome of the butterfly Papilio xuthus (Lepidoptera: Papilionidae) was sequenced for its nucleotide sequence of 13,964 bp. The genome has a typical gene order identical to other lepidopteran species. All tRNAs showed same stable canonical clover-leaf structure as those of other insects, except for tRNA(Ser) (AGN), in which the dihydrouracil arm (DHU arm) could not form stable stem-loop structure. Anomalous initiation codons have been observed for the cox1 gene, where the ATTACG hexa-nucleotide was believed to be involved in the initiation signaling. Twelve mitochondrial protein-coding gene sequence data were used to infer the phylogenetic relationships among the insect orders. Even though the number of insect orders represented by complete mitochondrial genomes is still limited, several well-established relationships are evident in the phylogenetic analysis of the complete sequences. Monophyly of the Homometabola was not supported in this paper. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, Papilionoidea and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea, Pyraloidea and Papilionoidea are monophyletic (Obtectomera). Bombycoidea (Bombyx mandarina and Antheraea pernyi) and Papilionoidea (P. xuthus and Coreana raphaelis) formed a sister group.

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.

Ommatidial type‐specific interphotoreceptor connections in the lamina of the swallowtail butterfly, Papilio xuthus

The eye of the butterfly Papilio xuthus contains a random array of three types of ommatidia (types I-III), each bearing nine photoreceptors, R1-R9. Of the six spectral classes of photoreceptors identified, types I, II, and III ommatidia contain four, three, and two classes, respectively: the ommatidia are thus spectrally heterogeneous. The photoreceptors send their axons to the lamina where, together with some large monopolar cells (LMCs), the nine from a single ommatidium contribute to a module called a lamina cartridge. We recently reported that among different photoreceptor axon terminals visualized by confocal microscopy, the number and length of axon collaterals differ for different spectral receptors, suggesting that lamina circuits are specific for each ommatidial type. Here we studied the distribution of synapse-like structures in the cartridges, first characterizing a photoreceptor by measuring its spectral sensitivity and then injecting Lucifer yellow (LY). We subsequently histologically identified the type of ommatidium to which the injected photoreceptor belonged, cut serial ultrathin sections of the entire lamina, labeled these with anti-LY immunocytochemistry, and then localized synapse-like structures. We found numerous interphotoreceptor contacts both within and between cartridges, the combination of which was again specific for the ommatidial type. R3 and R4, which are green-sensitive photoreceptors in all ommatidia, have thick axons lacking collaterals. We found that these cells exclusively make contacts with LMCs and not with photoreceptors. We therefore presume that R3 and R4 construct a system for motion vision, whereas other randomly distributed spectral types provide inputs for color vision.

Photoreceptor projection reveals heterogeneity of lamina cartridges in the visual system of the Japanese yellow swallowtail butterfly,Papilio xuthus

The compound eye of the butterfly Papilio xuthus is composed of three types of spectrally heterogeneous ommatidia. The ommatidia, which contain nine photoreceptor cells, R1-9, bear four (type I), three (type II), or two (type III) classes of spectral receptors in fixed combinations. The photoreceptors send their axons to the lamina, the first optic ganglion, where the R1-9 axons originating from a single ommatidium, together with some second-order neurons, form a neuronal bundle, called a lamina cartridge. We investigated the axonal structure of photoreceptors in the lamina to determine whether the cartridge structure is different between the three ommatidial types. We first characterized a photoreceptor by measuring its spectral sensitivity and then injected Lucifer Yellow. We subsequently identified the type of ommatidium of the injected photoreceptor via histological sections. We further observed the axonal structure of the photoreceptor in the lamina by laser confocal microscopy. We found that the number and length of axon collaterals markedly differ between the spectral receptors. Those having the most extensive axon collaterals, which extend into six or more surrounding cartridges, are violet receptors (R1 and R2 of type II ommatidia). UV receptors (R1 or R2 of type I ommatidia) also send collaterals into two to four neighboring cartridges. Blue receptors (R1 or R2 of type I ommatidia, R1 and R2 of type III ommatidia) have short collaterals restricted to their own cartridges. We thus conclude that the neuronal circuit of the lamina cartridge differs between the three types of ommatidia.

The role of wing veins in colour pattern development in the butterfly Papilio xuthus (Lepidoptera: Papilionidae)

Naturally occurring veinless specimen of the swallowtail Papilio xuthus show an extremely aberrant colour pattern. In spite of the fact that we have no breeding data, these veinless specimen are provisionally called veins-reduced mutant. In these mutants seven longitudinal veins of the fore wing and five of the hind wing are absent. The absence of wing veins is associated with a loss of the broad black venous stripes that normally are present along the proximal portion of the veins. In addition, missing veins cause a loss of the dislocation of black bands in adjacent wing cells, so that what are discrete black segments in normal wings become continuous bands in the veinless wing. Computer simulations show that the morphology of the striped patterns on both the veinless and veined wing can be explained if the wing margin acts as an inductive source of pattern formation and the veins act simply as boundaries to the propagation of the signal from the wing margin. The vein-dependent patterns by contrast, require that the veins act as inductive sources, at least along their proximal portion. This dual role of wing veins is consistent with prior observations on the biology of colour pattern formation. The unique veinless colour pattern strongly supports the hypothesis that the wing margin is the dominant organiser of colour pattern in this species, and possibly in other Papilionidae.

Colour constancy in the swallowtail butterfly Papilio xuthus.

We have recently shown that the Japanese yellow swallowtail butterfly Papilio xuthus uses colour vision when searching for food. In the field, these butterflies feed on nectar provided by flowers of various colours not only in direct sunlight but also in shaded places and on cloudy days, suggesting that they have colour constancy. Here, we tested this hypothesis. We trained newly emerged Papilio xuthus to feed on sucrose solution on a paper patch of a certain colour under white illumination. The butterflies were then tested under both white and coloured illumination. Under white illumination, yellow- and red-trained butterflies selected the correctly coloured patch from a four-colour pattern and from a colour Mondrian collage. Under four different colours of illumination, we obtained results that were fundamentally similar to those under white illumination. Moreover, we performed critical tests using sets of two similar colours, which were also correctly discriminated by trained butterflies under coloured illumination. Taken together, we conclude that the butterfly Papilio xuthus exhibits some degree of colour constancy when searching for food.

Tuning of Photoreceptor Spectral Sensitivities by Red and Yellow Pigments in the Butterfly Papilio xuthus

The compound eye of the Japanese yellow swallowtail butterfly, Papilio xuthus, consists of different types of ommatidia characterized by the pigmentation around the rhabdom. About 75% of the ommatidia harbor red pigment, whereas the other 25% contain yellow pigment. We find that the pigments function as spectral filters for the proximal photoreceptor cells. Intracellular recordings of the proximal cells yielded spectral sensitivities peaking in the red (λmax = 600 nm) and in the green (λmax = 520 nm), respectively. Staining of the recorded cells and subsequent histology demonstrated that the red receptors contain red pigment and that the green receptors contain yellow pigment. The sensitivity spectrum of the red receptors was considerably narrower compared to the absorption spectrum of a visual pigment peaking at 600 nm. The sensitivity spectrum can be calculated with an optical model for the butterfly rhabdom by incorporating measured absorbance spectra of the red pigments, yielding that the cell contains a visual pigment peaking at about 575 nm. The model also indicated that the spectral sensitivity of the green receptors is produced by the combination of the yellow lateral filter and a visual pigment peaking at 515 nm.

Effects of Sucrose Intake on Spermatophore Mass Produced by Male Swallowtail Butterfly Papilio xuthus L

Abstract A one-day old virgin male of the swallowtail butterfly, Papilio xuthus, requires about 50 min (at ca. 26.5°C) to transfer a spermatophore into the bursa copulatrix of a virgin female. Under such conditions, the spermatophore mass averages 4.7 mg, but is much smaller at the male's second mating (about a half). The energy stored during the larval stage is only enough to produce 1.5 spermatophores of the average size of the first mating. There is a positive correlation between adult male sugar intake and spermatophore mass at the second mating. To produce a spermatophore in a second mating that is the size typically produced by virgin males, a male must ingest 390 mg of sucrose solution (20%), which would take an estimated 4.3 days. If a large spermatophore is advantageous for mating, males are expected to have a period after mating when they feed but do not search for mates.

Hindsight by genitalia: photo-guided copulation in butterflies

1. Butterflies have two pairs of extraocular photoreceptive neurons on the genitalia. Here we report that the genital photoreceptors have a crucial role in achieving copulation. 2. We first investigated mating behavior of the butterfly Papilio xuthus in an outdoor cage. The mating behavior consists of six steps: the male approaches the female (1), maneuvers into a ventor-to-ventor position with the female (2), searches the female's genitalia (3), clasps the genitalia (4), the pair copulates (5), and finally separates (6). 3. We bilaterally ablated the P1 pair of photoreceptors, in the males, and observed mating behavior with virgin females. Of the intact males 66% copulated successfully, whereas only about 25% of the treated males could copulate. About 40% of P1 ablated males stopped the mating behavior during step 3: the males could not locate the female's genitalia. P1 ablation in females did not have a clear effect in this study. 4. Electrophysiological measurements showed that the P1 response of a male drops sharply when it correctly locates the female's genitalia. We hypothesize that the sharp drop in the P1 response informs the male that the female's vagina is correctly positioned for penis insertion. The P1 ablated males never experience such␣a␣response drop, as there is no P1 response to begin with.

Selective UV-induced damage to short-wavelength receptor cells in the butterflyPapilio xuthus

A technique that allows the specific inactivation of short-wavelength-sensitive photoreceptive cells has been needed for a long time. Such a technique could be useful in studies on the role(s) of UV-receptors in circadian rhythmicity, recognition of floral patterns, homing behaviour, and mate selection in arthropods. We provide ultrastructural evidence that short-wavelength receptor cells can be selectively damaged without affecting other spectral-cell types. Since the method does not require the killing of the experimental animal, the latter can be used in behavioural or other follow-up tests.

Termination profiles of photoreceptor cells in the larval eye of the swallowtail butterfly.

Profiles of the axonal endings of seven retinular cells (R1-7) from each of six stemmata (I-VI) of the butterfly Papilio xuthus were examined. Their axons enter the brain anteroventrally and project dorsally along the brain's lateral surface before entering the optic neuropil. Larval optic neuropil has two distinct areas, a (distal) lamina and a (proximal) medulla, connected by a chiasma. Intracellular administration of cobalt shows that distal retinular cells (R1-3 of stemmata I-IV, R1-3 and R6 of stemmata V and VI) terminate as short axons with plug or toothbrush-like endings in the lamina. Proximal retinular cells (R4-7 of stemmata I-IV, R4, R5 and R7 of stemmata V and VI) terminate as long axons in the chiasma or in the medulla. The long axons have fine branches in the lamina, chiasma and medulla. The two proximal cells (R5 and R7) are distinguished by their deeper endings and longer branches. The terminals of photoreceptors correspond to their photoreceptive properties and provide further evidence for the functional specialization of the photoreceptors.

Trophospongium-like structures in an insect eye: response of retinula cells of Papilio xuthus (Lepidoptera) to irradiation with ultraviolet light.

Presumably, as a consequence of a 3-h exposure to light of 350-nm wavelength (1.5 X 10(15) photons/cm2 sec) followed by a period of 20 h of rest in the dark, the four smaller retinula cells in each ommatidium of the eye of the butterfly Papilio xuthus contain a structure in the peripheral regions of their cytoplasm that resembles a portion of a rhabdom. Evidence for and against the view that this unusual, highly ordered arrangement of membranes represents a trophospongium is presented. In view of the fact that the structure in question only occurred at the same time when rhabdomeres were in a process of disintegration or reformation, the authors conclude that the structure in question is involved in the supply or removal of substances during a period of considerable activity of the retinula cell.