Neural control of tuneable skin iridescence in squid

Fast dynamic control of skin coloration is rare in the animal kingdom, whether it be pigmentary or structural. Iridescent structural coloration results when nanoscale structures disrupt incident light and selectively reflect specific colours. Unlike animals with fixed iridescent coloration (e.g. butterflies), squid iridophores (i.e. aggregations of iridescent cells in the skin) produce dynamically tuneable structural coloration, as exogenous application of acetylcholine (ACh) changes the colour and brightness output. Previous efforts to stimulate iridophores neurally or to identify the source of endogenous ACh were unsuccessful, leaving researchers to question the activation mechanism. We developed a novel neurophysiological preparation in the squid Doryteuthis pealeii and demonstrated that electrical stimulation of neurons in the skin shifts the spectral peak of the reflected light to shorter wavelengths (greater than 145 nm) and increases the peak reflectance (greater than 245%) of innervated iridophores. We show ACh is released within the iridophore layer and that extensive nerve branching is seen within the iridophore. The dynamic colour shift is significantly faster (17 s) than the peak reflectance increase (32 s), revealing two distinct mechanisms. Responses from a structurally altered preparation indicate that the reflectin protein condensation mechanism explains peak reflectance change, while an undiscovered mechanism causes the fast colour shift.     

Ultrastructure of the dermal chromatophores in a lizard (Scincidae: Plestiodon latiscutatus) with conspicuous body and tail coloration

Microscopic observation of the skin of Plestiodon lizards, which have body stripes and blue tail coloration, identified epidermal melanophores and three types of dermal chromatophores: xanthophores, iridophores, and melanophores. There was a vertical combination of these pigment cells, with xanthophores in the uppermost layer, iridophores in the intermediate layer, and melanophores in the basal layer, which varied according to the skin coloration. Skin with yellowish-white or brown coloration had an identical vertical order of xanthophores, iridophores, and melanophores, but yellowish-white skin had a thicker layer of iridophores and a thinner layer of melanophores than did brown skin. The thickness of the iridophore layer was proportional to the number of reflecting platelets within each iridophore. Skin showing green coloration also had three layers of dermal chromatophores, but the vertical order of xanthophores and iridophores was frequently reversed. Skin showing blue color had iridophores above the melanophores. In addition, the thickness of reflecting platelets in the blue tail was less than in yellowish-white or brown areas of the body. Skin with black coloration had only melanophores.     

The Blue Coloration of the Common Surgeonfish, Paracanthurus hepatus-II. Color Revelation and Color Changes

Measurements of spectral reflectance from the sky-blue portion of skin from the common surgeonfish, Paracanthurus hepatus, showed a relatively steep peak at around 490 nm. We consider that a multilayer thin-film interference phenomenon of the non-ideal type, which occurs in stacks of very thin light-reflecting platelets in iridophores of that region, is primarily responsible for the revelation of that hue. The structural organization of the iridophore closely resembles that of bluish damselfish species, although one difference is the presence of iridophores in a monolayer in the damselfish compared to the double layer of iridophores in the uppermost part of the dermis of surgeonfish. If compared with the vivid cobalt blue tone of the damselfish, the purity of the blue hue of the surgeonfish is rather low. This may be ascribable mainly to the double layer of iridophores in the latter since incident lightrays are complicatedly reflected and scattered in the strata. The dark-blue hue of the characteristic scissors-shaped pattern on the trunk of surgeonfish is mainly due to the dense population of melanophores, because iridophores are only present there in a scattered fashion. Photographic and spectral reflectance studies in vivo, as well as photomicrographic, photo-electric, and spectrometric examinations of the state of chromatophores in skin specimens in vitro, indicate that both melanophores and iridophores are motile. Physiological analyses disclosed that melanophores are under the control of the sympathetic nervous and the endocrine systems, while iridophores are regulated mainly by nerves. The body color of surgeonfish shows circadian changes, and becomes paler at night; this effect may be mediated by the pineal hormone, melatonin, which aggregates pigment in melanophores.     

Divisionistic generation of skin hue and the change of shade in the scalycheek damselfish, Pomacentrus lepidogenys

In addition to melanophores and xanthophores, there existed two types of iridophore in the dermis of the scalycheek damselfish, Pomacentrus lepidogenys. There are dendritic iridophores which reflect white light-rays by Tyndall scattering, and the round or somewhat ellipsoidal iridophores which reflect rays with a relatively narrow spectral peak from blue to green through the non-ideal thin-film interference. Most of the dendritic iridophores were covered with xanthophores and were situated over melanophores, thus constituting a kind of chromatophore unit which produces a yellow or yellowish-green color. The characteristic yellowish-green hue of the integument results from a compound effect of small contributions by more elementary colors. During color changes of the skin, the position of the spectral peak does not shift. Unlike the iridophores of the blue damselfish, both types of iridophore of the scalycheek damselfish were found to be inactive. It appears, therefore, that the aggregation and dispersion of pigment within the melanophores is the primary mechanism responsible for the changes in color of this species.     

Physiological color change in squid iridophores

Summary Cephalopods generally are thought to have only static iridophores, but this report provides qualitative and quantitative evidence for active control of certain iridescent cells in the dermis of the squidLolliguncula brevis. In vivo observations indicate the expression of iridescence to be linked to agonistic or reproductive behavior. The neuromodulator acetylcholine (ACh) induced dramatic optical changes in active iridophores in vitro, whereas ACh had little effect on passive iridophores elsewhere in the mantle skin. Bath application of physiological concentrations of ACh (10^-7M to 10^-6M) to excised dermal skin layers transformed the active iridophores from a non-reflective diffuse blue to brightly iridescent colors, and this reaction was reversible and repeatable. The speed of change to iridescent in vitro corresponded well to the speed of changes in the living animal. Pharmacological results indicate the presence of muscarinic receptors in this system and that Ca⁺⁺ is a mediator for the observed changes. Although ACh is present in physiological quantities in the dermal iridophore layer, it is possible that ACh release is not controlled directly by the nervous system because electrophysiological stimulation of major nerves in the periphery resulted in no iridescence inL. brevis; nor did silver staining or transmission electron microscopy reveal neuronal elements in the iridophore layer. Thus, active iridophores may be controlled by ACh acting as a hormone.     

Physiological color change in squid iridophores

Evidence is presented that changes in the optical properties of active iridophores in the dermis of the squid Lolliguncula brevis are the result of changes in the ultrastructure of these cells. At least two mechanisms may be involved when active cells change from non-iridescent to iridescent or change iridescent color. One is the reversible change of labile, detergent-resistant proteinaceous material within the iridophore platelets, from a contracted gel state (non-iridescent) to an expanded fluid or sol state when the cells become iridescent. The other is a change in the thickness of the platelets, with platelets becoming significantly thinner as the optical properties of the iridophores change from non-iridescent to iridescent red, and progressively thinner still as the observed iridescent colors become those of shorter wavelengths. Optical change from Rayleigh scattering (non-iridescent) to structural reflection (iridescent) may be due to the viscosity change in the platelet material, with the variations in observed iridescent colors due to changes in the dimensions of the iridophore platelets.     

Motile iridophores of a freshwater goby,Odontobutis obscura

Summary Reflecting chromatophores in the dermis of the skin of a freshwater goby, Odontobutis obscura, are of an iridophore type. These chromatophores contain numerous reflecting platelets, which are similar to those in iridophores of other fish and amphibian species. It was found that these iridophores are motile, i.e., these cells respond to certain stimuli with translocation of the platelets within the cells. K⁺ ions induced dispersion of the platelets in excised scale preparations, but not in excised scales from chemically denervated fish. Norepinephrine and melatonin also induced dispersion of the platelets. Alpha-MSH was effective in aggregating these organelles into the centrospheres of the cells. The conclusions reached are: (1) iridophores of O. obscura are motile; (2) the movement of the iridophores is under nervous and hormonal control.     

A Transmission Electron Microscopic (TEM) Method for Determining Structural Colors Reflected by Lizard Iridophores

Iridescent tissue colors are thought to be produced by iridophores through the optical phenomenon of thin-layer interference. Land and others have shown that structural features, predominantly reflecting platelet width and the cytoplasmic spacing between layers of platelets, determine the wavelength of light maximally reflected by this mechanism in iridophores. Some researchers have used interference microscopy to estimate these structural parameters, but the most direct measurement technique should be transmission electron microscopy (TEM). Transmission electron microscopy (TEM) has associated processing artifacts (particularly cytoplasmic shrinkage) that preclude direct measurement of ultrastructure, but if a number of assumptions are made, reflected wave-lengths can be predicted. A thin-layer interference model and its associated assumptions were tested using TEM measurements of iridophores from several brightly colored tissues of each of three lizards (Sceloporus jarroui, S. undulatus erythrocheilus, and S. magister). In all the instances examined when the contribution of the pigments present were accounted for, tissue color corresponded with predicted iridophore reflectances from the model. Finally, if the model and its assumptions are assumed to be correct, the amount of iridophore cytoplasmic shrinkage as a result of TEM processing can be calculated.     

Precise colocalization of interacting structural and pigmentary elements generates extensive color pattern variation in <Emphasis Type="Italic">Phelsuma</Emphasis>lizards

Background

Color traits in animals play crucial roles in thermoregulation, photoprotection, camouflage, and visual communication, and are amenable to objective quantification and modeling. However, the extensive variation in non-melanic pigments and structural colors in squamate reptiles has been largely disregarded. Here, we used an integrated approach to investigate the morphological basis and physical mechanisms generating variation in color traits in tropical day geckos of the genus Phelsuma.

Results

Combining histology, optics, mass spectrometry, and UV and Raman spectroscopy, we found that the extensive variation in color patterns within and among Phelsuma species is generated by complex interactions between, on the one hand, chromatophores containing yellow/red pteridine pigments and, on the other hand, iridophores producing structural color by constructive interference of light with guanine nanocrystals. More specifically, we show that 1) the hue of the vivid dorsolateral skin is modulated both by variation in geometry of structural, highly ordered narrowband reflectors, and by the presence of yellow pigments, and 2) that the reflectivity of the white belly and of dorsolateral pigmentary red marks, is increased by underlying structural disorganized broadband reflectors. Most importantly, these interactions require precise colocalization of yellow and red chromatophores with different types of iridophores, characterized by ordered and disordered nanocrystals, respectively. We validated these results through numerical simulations combining pigmentary components with a multilayer interferential optical model. Finally, we show that melanophores form dark lateral patterns but do not significantly contribute to variation in blue/green or red coloration, and that changes in the pH or redox state of pigments provide yet another source of color variation in squamates.

Conclusions

Precisely colocalized interacting pigmentary and structural elements generate extensive variation in lizard color patterns. Our results indicate the need to identify the developmental mechanisms responsible for the control of the size, shape, and orientation of nanocrystals, and the superposition of specific chromatophore types. This study opens up new perspectives on Phelsuma lizards as models in evolutionary developmental biology.

     

Adaptations of the reed frogHyperolius viridiflavus (Amphibia,Anura, Hyperoliidae) to its arid environment

Summary Of all amphibians living in arid habitats, reed frogs (belonging to the super speciesHyperolius viridiflavus) are the most peculiar. Froglets are able to tolerate dry periods of up to 35 days or longer immediately after metamorphosis, in climatically exposed positions. They face similar problems to estivating juveniles, i.e. endurance of long periods of high temperature and low RH with rather limited energy and water reserves. In addition, they must have had to develop mechanisms to prevent poisoning by nitrogenous wastes that rapidly accumulate during dry periods as a metabolic consequence of maintaining a non-torpid state.During dry periods, plasma osmolarity ofH. v. taeniatus froglets strongly increased, mainly through urea accumulation. Urea accumulation was also observed during metamorphic climax.During postmetamorphic growth, chromatophores develop with the density and morphology typical of the adult pigmentary pattern. The dermal iridophore layer, which is still incomplete at this time, is fully developed within 4–8 days after metamorphosis, irrespective of maintenance conditions. These iridophores mainly contain the purines guanine and hypoxanthine. The ability of these purines to reflect light provides an excellent basis for the role of iridophores in temperature regulation. In individuals experiencing dehydration stress, the initial rate of purine synthesis is doubled in comparison to specimens continuously maintained under wet season conditions. This increase in synthesis rate leads to a rapid increase in the thickness of the iridophore layer, thereby effectively reducing radiation absorption. Thus, the danger of overheating is diminished during periods of water shortage when evaporative cooling must be avoided. After the development of an iridophore layer of sufficient thickness for effective radiation reflectance, synthesis of iridophore pigments does not cease. Rather, this pathway is further used during the remaining dry season for solving osmotic problems caused by accumulation of nitrogenous wastes. During prolonged water deprivation, in spite of reduced metabolic rates, purine pigments are produced at the same rate as in wet season conditions. This leads to a higher relative proportion of nitrogen end products being stored in skin pigments under dry season conditions. At the end of an experimental dry season lasting 35 days, up to 38% of the accrued nitrogen is stored in the form of osmotically inactive purines in the skin. Thus the osmotic problems caused by evaporative water loss and urea production are greatly reduced.     

FMRFamide elicits chromatophore expansion and retraction depending on its type and development in the squid, Sepioteuthis lessoniana

To examine chromatophore control by FMRFamide-related peptide (FaRP), we investigated the pharmacological effect of FMRFamide on the chromatophores and the FMRFamide-immunoreactivity of nerves surrounding the muscles in the coastal squid, Sepioteuthis lessoniana. Applications of FMRFamide elicited expansion of black chromatophores and retraction of yellow chromatophores in the adult squid. FMRFamide-immunoreactive terminals were distributed along black chromatophore muscles but were not observed around the yellow ones. This means that FMRFamide functions differently for each of the two types of chromatophores in the adult squid. Moreover, the pharmacological effect of FMRFamide on the black chromatophores differed between adults and hatchlings; application of FMRFamide retracted black chromatophores in hatchlings but not in adults. These results indicate that certain squid species have an FaRP system for controlling the chromatophores in their skin and that the system changes during development.     

Stress‐induced changes in color expression mediated by iridophores in a polymorphic lizard

Stress is an important potential factor mediating a broad range of cellular pathways, including those involved in condition‐dependent (i.e., honest) color signal expression. However, the cellular mechanisms underlying the relationship between stress and color expression are largely unknown. We artificially elevated circulating corticosterone levels in male tawny dragon lizards, Ctenophorus decresii, to assess the effect of stress on the throat color signal. Corticosterone treatment increased luminance (paler throat coloration) and decreased the proportion of gray, thereby influencing the gray reticulations that produce unique patterning. The magnitude of change in luminance for corticosterone‐treated individuals in our study was around 6 “just noticeable differences” to the tawny dragon visual system, suggesting that lizards are likely to be able to perceive the measured variation. Transmission electron microscopy (TEM) of iridophore cells indicated that luminance increased with increasing density of iridophore cells and increased spacing (and/or reduced size) of crystalline guanine platelets within them. Crystal spacing within iridophores also differed between skin colors, being greater in cream than either gray or yellow skin and greater in orange than yellow skin. Our results demonstrate that stress detectably impacts signal expression (luminance and patterning), which may provide information on individual condition. This effect is likely to be mediated, at least in part, by structural coloration produced by iridophore cells.     

Reflecting elements in cephalopod skin and their importance for camouflage

Attention is drawn to the fact that cephalopod skin contains reflecting elements (irido-phores, leucophores) in addition to the well-known chromatophores; and the reflexion of monochromatic light from small areas all over the arm, head and body skin in Octopus vulgaris and Sepia officinalis is demonstrated. It is emphasized that the chromatophores of these species contain pigments only at the yellow-red (-black) end of the spectrum so that no combination of their activities could cause the skin to take on the blue or green tinge occasionally seen in the living animal. The function of the chromatophores in cryptic "colouration" is largely limited to tone-matching, colour-matching being dealt with by the reflecting elements. These, when they are revealed by retraction of the overlying chromatophores, automatically reflect incident light of whatever wavelength and thus enable what is probably a colour-blind animal to take on the colour as well as the tone of its surroundings.     

The effect of colour of the substrate on the landing and oviposition behaviour of the cabbage root fly

Field and laboratory experiments were made in order to understand the relation between the spectral characteristic of a substrate and its attractiveness for Delia radicum (L.) (Diptera: Anthomyiidae) flies to land or oviposit. Landing females preferred substrates with high reflectance of green and particularly of yellow wavelengths, but substrates with a high proportion of the blue and green reflectance simultaneously (light blue and white) were also very attractive. Unattractive substrates had either low reflectance across the whole insect-visible spectrum (dark blue) or increased reflectance only in orange or red region of the spectrum (red) or a large proportion of UV reflectance (aluminium). Landing males were most attracted to the substrates with the highest total reflection. Oviposition attractiveness of a substrate grew with an increase in the proportion of reflectance in the blue and a decrease in the green regions of the spectrum. In addition, the oviposition attractiveness increased with increasing total reflection and contrast with the background, and decreased with a high proportion of UV reflectance.     

On leucophores and the chromatic unit of Octopus vulgaris

The leucophores of Octopus vulgaris are much branched cells bearing, all over the surface, small clubs that contain electron-dense material of an unknown nature. Some tens of leucophores are organized to form a "sponge" in the centre of the chromatic unit, which can be elevated above the peripheral region. This central region reflects light over a broad band and may be important in camouflage.
The chromatic unit also contains iridophores and chromatophores. The latter may be yellow, orange, red or black, and the different types are interspersed among one another in a regular manner. The chromatophores are disposed in a precise way with respect to the leucophores, the black ones lying above holes in the sponge. The possible significance of this arrangement for camouflage is discussed.     

Rapid integumental color changes due to novel iridophores in the chameleon sand tilefish Hoplolatilus chlupatyi

The wavelength of the light reflected from iridophores depends on the thickness and the spacing of intracellular reflecting platelets. Here, we show that the rapid color change from blue to red of the chameleon sand tilefish Hoplolatilus chlupatyi is mediated by adrenergic stimulation of a novel type of iridophore in which reflecting platelets are concentrated selectively in the periphery of the cell, near the plasma membrane. The color changes are not only observed in vivo but also in pigment cells of isolated scales which respond to increases in K⁺ ion concentrations in 0.5 s and to addition of norepinephrine within 1 s. The norepinephrine effect can be blocked by addition of the alpha‐adrenergic antagonist phentolamine. The results suggest that adrenergic stimulation leads to changes in reflecting platelet organization in Hoplolatilus chlupatyi iridophores and represents the major mediator of the rapid color change in this fish in vivo.     

Hormone induced chromatophore changes in the European tree frog, Hyla arborea, in vitro

The colours of the European tree frog, Hvlu urhorea , depend on three types of chromatophores: in dermo-epidermal direction melanophores, iridophores, and xanthophores. The ability ofthis species to assume a wide range ofcolours implies that very extensive changes in the chromatophores take place, which in turn require control by several regulating factors. The responses of the different chromatophore types to hormones with known melanophore-affecting abilities (α-MSH, β-MSH, ACTH, melatonin) were tested in an in vitro system (freshly explanted skin) using reflectance microspectrophotometry, light microscopy and time-lapse cinemicrography.
α-MSH, β-MSH and ACTH all induce a rapid dispersion of melanosomes during the 10 min after addition. The degree of pigment dispersion induced by ACTH is slightly less than after stimulation with α-MSH or β-MSH.
The iridophores react to MSH or ACTH treatment with a contraction of the entire cell (causing a reduction in reflecting area), and a change in orientation of the platelets, causing a decrease in selective reflectance. The iridophores appear to be especially sensitive to ACTH. A very striking feature of the iridophores when studied with time-lapse cinematography is their strong pulsations (approx. once per minute).
The xanthophores react to MSH and ACTH with a contraction. These cells appear to be sensitive to β-MSH in particular.
Melatonin strongly counteracts the effects of α-MSH, β-MSH and ACTH on all chromatophores.
These studies confirm the dynamic nature not only of the melanophores, but also of the iridophores and xanthophores, as pointed out by Schmidt (1920) and Nielsen (1978a). Furthermore the differences in the time course of the stimulation of the different types of chromatophores by various hormones may provide an experimental basis for the explanation of colour changes in Hyfa arboreu.     

Use of CC traps with different trap base colors for silverleaf whiteflies (Homoptera: Aleyrodidae), thrips (Thysanoptera: Thripidae), and leafhoppers (Homoptera: Cicadellidae)

During 1996, 1997, and 1999, studies were conducted in cotton, sugar beets, alfalfa, yardlong bean, and peanut fields to compare insect catches in CC traps equipped with different trap base colors. The studies were conducted in southwestern United States, China, and India. The nine colors, white, rum, red, yellow, lime green, spring green, woodland green (dark green), true blue, and black, varied in spectral reflectance in the visible (400-700 nm) and near-infrared (700-1050 nm) portions of spectrum. Lime green, yellow, and spring green were the three most attractive trap base colors for silverleaf whitefly, Bemisia argentifolii Bellows & Perring, and leafhopper, Empoasca spp. adults. The three trap base colors were moderately high in the green, yellow, and orange spectral regions (490-600 nm), resembling the spectral reflectance curve of the abaxial (underleaf) surfaces of green cotton leaves. True blue and white were the most attractive trap base colors for western flower thrips, Frankliniella occidentalis (Pergande), adults. The true blue and white trap bases were moderately high in the blue spectral region (400-480 nm).     

Color matching on natural substrates in cuttlefish, <Emphasis Type="Italic">Sepia officinalis</Emphasis>

The camouflaging abilities of cuttlefish (Sepia officinalis) are remarkable and well known. It is commonly believed that cuttlefish-although color blind-actively match various colors of their immediate surroundings, yet no quantitative data support this notion. We assembled several natural substrates chosen to evoke the three basic types of camouflaged body patterns that cuttlefish express (uniform/stipple, mottle, and disruptive) and measured the spectral reflectance of the camouflaged pattern and the respective background using a fiber optic spectrometer. We demonstrate that the reflectance spectra of cuttlefish skin patterns correlate closely with the spectra of these natural substrates. Since pigmented chromatophores play a key role in cephalopod color change, we also measured the spectral reflectance of individual cuttlefish chromatophores under the microscope, and confirm the results from a previous publication reporting three distinct colors of chromatophores (yellow, orange, and dark brown) on the animals' dorsal side. Taken together, our results show that the color variations in substrate and animal skin can be very similar and that this may facilitate color match on natural substrates in the absence of color vision.