Atsuko Matsushita

Extreme Spectral Richness in the Eye of the Common Bluebottle Butterfly, Graphium sarpedon

Butterfly eyes are furnished with a variety of photoreceptors of different spectral sensitivities often in species-specific manner. We have conducted an extensive comparative study to address the question of how their spectrally complex retinas evolved. Here we investigated the structure and function of the eye of the common bluebottle butterfly (Graphium sarpedon), using electrophysiological, anatomical and molecular approaches. Intracellular electrophysiology revealed that the eye contains photoreceptors of fifteen distinct spectral sensitivities. These can be divided into six spectral receptor classes: ultraviolet- (UV), violet- (V), blue- (B), blue-green- (BG), green- (G), and red- (R) sensitive. The B, G and R classes respectively contain three, four and five subclasses. Fifteen is the record number of spectral receptors so far reported in a single insect eye. We localized these receptors by injecting dye into individual photoreceptors after recording their spectral sensitivities. We thus found that four of them are confined to the dorsal region, eight to the ventral, and three exist throughout the eye; the ventral eye region is spectrally richer than the dorsal region. We also identified mRNAs encoding visual pigment opsins of one ultraviolet, one blue and three long wavelength-absorbing types. Localization of these mRNAs by in situ hybridization revealed that the dorsal photoreceptors each express a single opsin mRNA, but more than half of the ventral photoreceptors coexpress two or three L opsin mRNAs. This expression pattern well explains the spectral organization of the Graphium compound eye.

Glass scales on the wing of the swordtail butterfly Graphium sarpedon act as thin film polarizing reflectors

SUMMARY The wings of the swordtail butterfly Graphium sarpedon (the Common Bluebottle) have blue/green-colored patches that are covered on the underside by two types of scales: white and glass scales. Transmission and scanning electron microscopy revealed that the white scales are classically structured: the upper lamina, with prominent ridges and large open windows, is well separated by trabeculae from a flat, continuous lower lamina. In the glass scales, the upper lamina, with inconspicuous ridges and windows, is almost flat and closely apposed to the equally flat lower lamina. The glass scales thus approximate ideal thin films, in agreement with the observation that they reflect light directionally and are iridescent. Reflectance and transmittance spectra measured from the glass scales with a microspectrophotometer agree with spectra calculated for an ideal non-absorbing thin film. Imaging scatterometry of single, isolated glass scales demonstrated that the reflected light can be strongly polarized, indicating that they function as polarizing reflectors.

Spectrally tuned structural and pigmentary coloration of birdwing butterfly wing scales

The colourful wing patterns of butterflies play an important role for enhancing fitness; for instance, by providing camouflage, for interspecific mate recognition, or for aposematic display. Closely related butterfly species can have dramatically different wing patterns. The phenomenon is assumed to be caused by ecological processes with changing conditions, e.g. in the environment, and also by sexual selection. Here, we investigate the birdwing butterflies, Ornithoptera, the largest butterflies of the world, together forming a small genus in the butterfly family Papilionidae. The wings of these butterflies are marked by strongly coloured patches. The colours are caused by specially structured wing scales, which act as a chirped multilayer reflector, but the scales also contain papiliochrome pigments, which act as a spectral filter. The combined structural and pigmentary effects tune the coloration of the wing scales. The tuned colours are presumably important for mate recognition and signalling. By applying electron microscopy, (micro-)spectrophotometry and scatterometry we found that the various mechanisms of scale coloration of the different birdwing species strongly correlate with the taxonomical distribution of Ornithoptera species.

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.

Neuronal Sensitivity to Microsecond Time Disparities in the Electrosensory System of Gymnarchus niloticus

To perform the jamming avoidance response (JAR), the weakly electric fish Gymnarchus detects time disparities on the order of microseconds between electrosensory signals received by electroreceptors in different parts of the body surface. This paper describes time-disparity thresholds of output neurons of the electrosensory lateral line lobe (ELL), where the representation of timing information is converted from a time code to a firing-rate code. We recorded extracellular single-unit responses from pyramidal cells in the ELL to sinusoidally modulated time disparity with various depths (0-200 μs). Threshold sensitivity to time disparities measured in 123 units ranged from 0.5 to 100 μs and was ≤5 μs in 60% of the units. The units from pyramidal cells in the inner and outer cell layers of the ELL responded equally well to small time disparities. The neuronal thresholds to time disparities found in the ELL are comparable with those demonstrated in behavioral performance of the JAR. The sensitivity of ELL units to small time disparities was unaffected when the center of the cyclic time-disparity modulation was shifted over a wide range (up to 250 μs), indicating an adaptation mechanism for steady-state time disparities that preserves the sensitivity to small dynamic changes in time disparities. Phase-locked input neurons, which provide time information to the ELL by phase-locked firing of action potentials, did not adapt to steady-state time shifts of sensory signals. This suggests that the adaptation emerges within the ELL.

Combined pigmentary and structural effects tune wing scale coloration to color vision in the swallowtail butterfly Papilio xuthus

Butterflies have well-developed color vision, presumably optimally tuned to the detection of conspecifics by their wing coloration. Here we investigated the pigmentary and structural basis of the wing colors in the Japanese yellow swallowtail butterfly, Papilio xuthus, applying spectrophotometry, scatterometry, light and electron microscopy, and optical modeling. The about flat lower lamina of the wing scales plays a crucial role in wing coloration. In the cream, orange and black scales, the lower lamina is a thin film with thickness characteristically depending on the scale type. The thin film acts as an interference reflector, causing a structural color that is spectrally filtered by the scale’s pigment. In the cream and orange scales, papiliochrome pigment is concentrated in the ridges and crossribs of the elaborate upper lamina. In the black scales the upper lamina contains melanin. The blue scales are unpigmented and their structure differs strongly from those of the pigmented scales. The distinct blue color is created by the combination of an optical multilayer in the lower lamina and a fine-structured upper lamina. The structural and pigmentary scale properties are spectrally closely related, suggesting that they are under genetic control of the same key enzymes. The wing reflectance spectra resulting from the tapestry of scales are well discriminable by the Papilio color vision system.

Eyes with basic dorsal and specific ventral regions in the glacial Apollo, Parnassius glacialis (Papilionidae)

SUMMARY Recent studies on butterflies have indicated that their colour vision system is almost species specific. To address the question of how this remarkable diversity evolved, we investigated the eyes of the glacial Apollo, Parnassius glacialis, a living fossil species belonging to the family Papilionidae. We identified four opsins in the Parnassius eyes – an ultraviolet- (PgUV), a blue- (PgB), and two long wavelength (PgL2, PgL3)-absorbing types – and localized their mRNAs within the retina. We thus found ommatidial heterogeneity and a clear dorso-ventral regionalization of the eye. The dorsal region consists of three basic types of ommatidia that are similar to those found in other insects, indicating that this dorsal region retains the ancestral state. In the ventral region, we identified two novel phenomena: co-expression of the opsins of the UV- and B-absorbing type in a subset of photoreceptors, and subfunctionalization of long-wavelength receptors in the distal tier as a result of differential expression of the PgL2 and PgL3 mRNAs. Interestingly, butterflies from the closely related genus Papilio (Papilionidae) have at least three long-wavelength opsins, L1–L3. The present study indicates that the duplication of L2 and L3 occurred before the Papilio lineage diverged from the rest, whereas L1 was produced from L3 in the Papilio lineage.

Not flying blind: A comparative study of photoreceptor function in flying and non-flying cockroaches

ABSTRACT Flying is often associated with superior visual performance, as good vision is crucial for detection and implementation of rapid visually guided aerial movements. To understand the evolution of insect visual systems it is therefore important to compare phylogenetically related species with different investments in flight capability. Here, we describe and compare morphological and electrophysiological properties of photoreceptors from the habitually flying green cockroach Panchlora nivea and the American cockroach Periplaneta americana, which flies only at high ambient temperatures. In contrast to Periplaneta, ommatidia in Panchlora were characterized by two-tiered rhabdom, which might facilitate detection of polarized light while flying in the dark. In patch-clamp experiments, we assessed the absolute sensitivity to light, elementary and macroscopic light-activated current and voltage responses, voltage-activated potassium (Kv) conductances, and information transfer. Both species are nocturnal, and their photoreceptors were similarly sensitive to light. However, a number of important differences were found, including the presence in Panchlora of a prominent transient Kv current and a generally low variability in photoreceptor properties. The maximal information rate in Panchlora was one-third higher than in Periplaneta, owing to a substantially higher gain and membrane corner frequency. The differences in performance could not be completely explained by dissimilarities in the light-activated or Kv conductances; instead, we suggest that the superior performance of Panchlora photoreceptors mainly originates from better synchronization of elementary responses. These findings raise the issue of whether the evolutionary tuning of photoreceptor properties to visual demands proceeded differently in Blattodea than in Diptera. Summary: Comparison of photoreceptors of flying Panchlora and non-flying Periplaneta cockroaches using imaging and electrophysiological methods reveals prominent differences, which are discussed in relation to the visual ecological paradigm developed for Diptera.

Disruption of actin filament organization by cytochalasin D inhibits rhabdom synthesis in the compound eye of the crab Hemigrapsus sanguineus

Abstract.This paper demonstrates that the actin filaments in the photoreceptor cell body of the crab Hemigrapsus sanguineus are involved in the transport of the smooth endoplasmic reticulum in the process of rhab- dom synthesis. In isolated eyes, the transverse sectional area of a rhabdom increased at dusk. The increase was inhibited by disrupting the actin filaments with cytochalasin D. In the photoreceptor cell body of cytochalasin D-treated eyes, rhodamine-conjugated phalloidin labeled some spots, which appeared to be the aggregates of short filaments as revealed by electron microscopy. The photoreceptor cell bodies in cytochalasin D-treated eyes contained more vesicular smooth endoplasmic reticulum than those in the control eye. These results indicate that the disruption of actin organization with cytochalasin D resulted in the accumulation of smooth endoplasmic reticulum. Microtubules do not play a direct role in the rhabdom synthesis because colchicine failed to stop the rhabdom increase.

Cholesterol homeostasis in the ovaries of neonatally diethylstilbestrol-treated mice.

In the ovary of neonatally DES-treated mice, lipid droplets accumulation was observed in the hypertrophied interstitial tissues. Our previous results demonstrated that the impaired steroidogenesis in the ovary of neonatally DES-treated mice was caused by altered gonadotropins levels, and resulted in the hypertrophy of ovarian interstitial cells. We speculated that lipid droplets in the ovary mainly consisted of cholesterol. This study was aimed to examine the effects of neonatal DES on cholesterol homeostasis in the ovary. The serum and ovarian total cholesterol concentrations in 3-month-old neonatally DES-treated mice were significantly higher than those in the neonatally oil-treated mice, but triglyceride concentrations were not altered. In the ovary of neonatally DES-treated mice, expression of Hmgcr, a rate-limiting enzyme in de novo cholesterol biosynthesis, was reduced but expression of Ldlr and Scarb1, involved in cholesterol uptake, was not changed. These results suggest that cholesterol uptake is not altered in the ovary of 3-month-old neonatally DES-treated mice. However, the expression of Acat1, the microsomal acyl coenzyme A cholesterol acyltransferase which is involved in cholesterol esterification and storing was increased compared with that in the ovary of neonatally oil-treated mice. Since ovarian steroidogenesis in neonatally DES-treated mice was impaired, synthesized and/or obtained cholesterol from the blood may not be used sufficiently. Thus, in the ovary of neonatally DES-treated mice, cholesterol is esterified by ACAT1 and stored in the interstitial cells.