Class Of Pigments Research Articles

Elucidating the Structural Framework of Synthetic and Fungal Melanins by Solid-State NMR

Melanins are an important class of natural pigments associated with biological functions that include protection from sunlight, camouflage, free radical scavenging, and microbial virulence. However, structural investigation of these enigmatic pigments remains a challenging task because of their insolubility and heterogeneous architecture. High-resolution solid-state NMR spectroscopy offers a unique tool to probe melanin structure, and melanogenesis in Cryptococcus neoformans (CN) requiring exogenous obligatory catecholamine precursors such as L-dopa provides unparalleled opportunities to investigate melanin assembly in fungal cells. 1D solid-state 13C NMR and EPR demonstrated the structural differences among CN melanins produced by systematic variation of catecholamine precursors and in cell-free media. Broad aromatic resonances in both fungal and synthetic melanins indicated a heterogeneous disordered aromatic domain in these pigments, whereas differences among the aliphatics suggested incorporation of glucose-derived cellular materials in the fungal materials. A mechanistic scheme was proposed based on the Mason-Raper pathway, including potential intermediates for the biosynthesis of indole-based melanin polymers and the consequent precursor preferences observed in CN melanogenesis. 13C and 15N solid-state NMR, including 2D through-space and through-bond correlation spectra of pigments produced with exogenously 13C-and 15N-enriched L-dopa and/or [U-13C6]-glucose supplied to the growth media, revealed the molecular framework formed by the pigments and in CN melanization. Findings include: (1) the major aromatic moieties are located spatially within 5-7 A of the long-chain aliphatics; (2) glucose scrambles metabolically to form oxygenated- and long-chain aliphatic structures; (3) particular aromatic, oxygenated- and long-chain aliphatic moieties are attached covalently to carboxyl groups of the pigment; (4) nitrogenous components are found primarily in the aromatic domain of both synthetic and fungal pigments. The structural framework of melanins deduced in this investigation can guide the design of new antivirulence drugs and therapeutics against melanoma tumors.

Spectral tuning of Amazon parrot feather coloration by psittacofulvin pigments and spongy structures

The feathers of Amazon parrots are brightly coloured. They contain a unique class of pigments, the psittacofulvins, deposited in both barbs and barbules, causing yellow or red coloured feathers. In specific feather areas, spongy nanostructured barb cells exist, reflecting either in the blue or blue-green wavelength range. The blue-green spongy structures are partly enveloped by a blue-absorbing, yellow-colouring pigment acting as a spectral filter, thus yielding a green coloured barb. Applying reflection and transmission spectroscopy, we characterized the Amazons' pigments and spongy structures, and investigated how they contribute to the feather coloration. The reflectance spectra of Amazon feathers are presumably tuned to the sensitivity spectra of the visual photoreceptors.

The effects of temperature and pH on stability of anthocyanins from red sorghum (Sorghum bicolor) bran

Anthocyanin’s have been suggested as promising dietary compounds with an important role in human health. This study focuses on the effect of temperature and pH on stability of anthocyanins from red sorghum bran. The sorghum anthocyanins were found to be stable at 0°C even when exposed to light and dark conditions. The anthocyanins extracted using acidified methanol showed higher stability than methanol extracts. The study on anthocyanin stability over the pH range (1.1 to 10.5) during 20 days storage was conducted on sorghum anthocyanins compared to methanol and acidified methanol extracts afforded higher colour intensity and higher or similar stability throughout the whole pH range. At pH 8.9, the colour intensity of sorghum anthocyanins was even higher than at the lowest pH values. The sorghum 3-deoxyanthocyanins were a rare class of plant pigments, and were stable to light, heat and change in pH. The use of sorghum anthocyanins as food colourants in slightly alkaline products (bakery, milk, egg, etc.) can therefore be considered. Key words: Sorghum, bran, anthocyanins, temperature, pH stability.

C–H activation: making diketopyrrolopyrrole derivatives easily accessible

Diketopyrrolopyrrole (DPP) derivatives are an important class of high-performance pigment used in inks, paints, plastics, and organic electronics. Until now, DPP derivatives containing sophisticated aryl units at the DPP core have usually been obtained via Suzuki, Stille, or Negishi cross-coupling reactions, which require organometallic precursors. In this work, a series of DPP-based π-conjugated molecules bearing diverse aryl substituents on the thiophene- or benzene-DPPs were facilely synthesized in moderate to excellent yields through the Pd-catalyzed direct arylation of C–H bonds. The synthetic procedures feature advantages over traditional C–C cross-coupling reactions such as: (1) avoidance of the use of organometallic reagents in the starting materials leading to simpler byproducts and higher atom economy, (2) fewer synthetic steps, (3) higher yields, (4) better compatibility with chemically sensitive functional groups, and (5) simpler catalytic systems free of phosphine ligands. These advantages make the present protocol an ideal and versatile strategy for the synthesis of DPP derivatives, especially for structurally complicated DPPs that may possess chemically sensitive functionalities. The optical and electrochemical properties of the synthesized DPPs (17 compounds) were systematically investigated using UV-vis spectroscopy, steady-state fluorescence spectroscopy, and cyclic voltammetry (CV).

Fisetin: A Dietary Antioxidant for Health Promotion

Diet-derived antioxidants are now being increasingly investigated for their health-promoting effects, including their role in the chemoprevention of cancer. In general, botanical antioxidants have received much attention, as they can be consumed for longer periods of time without any adverse effects. Flavonoids are a broadly distributed class of plant pigments that are regularly consumed in the human diet due to their abundance. One such flavonoid, fisetin (3,3',4',7-tetrahydroxyflavone), is found in various fruits and vegetables, such as strawberry, apple, persimmon, grape, onion, and cucumber. Several studies have demonstrated the effects of fisetin against numerous diseases. It is reported to have neurotrophic, anticarcinogenic, anti-inflammatory, and other health beneficial effects. Although fisetin has been reported as an anticarcinogenic agent, further in-depth in vitro and in vivo studies are required to delineate the mechanistic basis of its observed effects. In this review article, we describe the multiple effects of fisetin with special emphasis on its anticancer activity as investigated in cell culture and animal models. Additional research focused toward the identification of molecular targets could lead to the development of fisetin as a chemopreventive/chemotherapeutic agent against cancer and other diseases.

Production of cerium zinc molybdate nano pigment by innovative ultrasound assisted approach

Ultrasound assisted synthesis of yellow rare earth cerium zinc molybdate anticorrosion nanopigment is presented. This new class of pigment is eco-friendly alternatives to lead, cadmium and chromium pigment as these pigments contains carcinogenic species like Cr6+ which is responsible for human disease. The synthesis of nanosized cerium zinc molybdate was carried out using cerium nitrate, sodium zinc molybdate as a precursor materials by conventional and ultrasound assisted chemical precipitation method without addition of emulsification agent. XRD, FTIR and elemental analysis confirm the formation of cerium zinc molybdate nanoparticles. The conductivity results indicate that conventional synthesis takes longer time, while in sonochemical technique (US), reaction completes within short period of time. Improved solute transfer rate, rapid nucleation, and formation of large number of nuclei are attributed to presence of cavitation. Saturation of the Ce3+ ions reaches earlier in case of sonochemical technique which restricts the growth of particles hence smaller size is obtained. The crystallite size of cerium zinc molybdate was found to be 27nm from XRD analysis.

Influence of exogenously applied abscisic acid on carotenoid content and water uptake in flowers of the tea plant (Camellia sinensis)

Carotenoids are a major class of plant pigments and fulfill many functions in different organisms that either produce or consume them. Although the color of the stamina of tea (Camellia sinensis) flowers is clearly due to the presence of carotenoids, the carotenoid profile and content remain to be discovered. We investigated the carotenoid profile of tea flowers and determined changes in concentrations over the floral development. The flowers contained oxygenated xanthophylls such as neoxanthin, lutein and zeaxanthin, as well as the hydrocarbons β-carotene and α-carotene. Flowers of the tea plant contain to vegetables comparable amounts of carotenoids. The content of 9'-cis-epoxycarotenoids, which serve as abscisic acid precursors, as well as changes in concentration of abscisic acid were studied. The concentrations of carotenoids decreased whereas the abscisic acid content increased over the floral development. Exogenously applied S-abscisic acid affected water uptake, flower opening and carotenoid accumulation. In summary, this paper reports, for the first time, the carotenoid profile and content of tea flowers. The study revealed that carotenoids in tea flowers are an interesting target in respect of possible applications of tea flower extracts as well as biological functions of abscisic acid during floral development.

Towards the development of a novel bioinspired functional material: Synthesis and characterization of hybrid TiO2/DHICA-melanin nanoparticles

A large number of recent literature data focus on modification/modulation of surface chemistry of inorganic materials in order to improve their functional properties. Melanins, a wide class of natural pigments, are recently emerging as a powerful organic component for developing bioinspired active material for a large number of applications from organoelectronics to bioactive compounds.Here we report the use of the approach referred as “chimie douce”, involving in situ formation of the hybrids through reactions of precursors under mild conditions, to prepare novel hybrid functional architectures based on eumelanin like 5,6 dihydroxyindole-2-carboxylic acid (DHICA) polymer and TiO2. Two synthesis procedures were carried out to get DHICA-melanin coated TiO2 nanoparticles as well as mixed DHICA/TiO2 hybrid nanostructures. Such systems were characterized through EPR, FT-IR and fluorescence spectroscopy, thermogravimetric analysis (TGA), X-ray diffraction (XRD), and TEM microscopy in order to assess the effect of synthesis path as well as of DHICA content on structural, morphological and optical properties of TiO2 nanostructures. In particular, EPR, FT-IR spectra and TGA analysis confirmed the presence of DHICA-melanin in these samples. TEM measurements indicated the formation of the nanoparticles having relatively narrow size distribution with average particle size of about 10nm. DHICA-melanin does act as a morphological agent affecting morphology of hybrid nanostructures. XRD analysis proved that TiO2 hybrid nanoparticles kept anatase structures for DHICA-melanin contents within the range of investigated compositions, i.e. up to 50% wt/wt.

Spectral tuning by selective chromophore uptake in rods and cones of eight populations of nine-spined stickleback (Pungitius pungitius)

The visual pigments of rods and cones were studied in eight Fennoscandian populations of nine-spined stickleback (Pungitius pungitius). The wavelength of maximum absorbance of the rod pigment (λ(max)) varied between populations from 504 to 530 nm. Gene sequencing showed that the rod opsins of all populations were identical in amino acid composition, implying that the differences were due to varying proportions of chromophores A1 and A2. Four spectral classes of cones were found (two S-cones, M-cones and L-cones), correlating with the four classes of vertebrate cone pigments. For quantitative estimation of chromophore proportions, we considered mainly rods and M-cones. In four populations, spectra of both photoreceptor types indicated A2 dominance (population mean λ(max)=525-530 nm for rods and 535-544 nm for M-cones). In the four remaining populations, however, rod spectra (mean λ(max)=504-511 nm) indicated strong A1 dominance, whereas M-cone spectra (mean λ(max)=519-534 nm) suggested substantial fractions of A2. Quantitative analysis of spectra by three methods confirmed that rods and cones in these populations use significantly different chromophore proportions. The outcome is a shift of M-cone spectra towards longer wavelengths and a better match to the photic environment (light spectra peaking >560 nm in all the habitats) than would result from the chromophore proportions of the rods. Chromophore content was also observed to vary partly independently in M- and L-cones with potential consequences for colour discrimination. This is the first demonstration that selective processing of chromophore in rods and cones, and in different cone types, may be ecologically relevant.

Using solid-state NMR to monitor the molecular consequences of Cryptococcus neoformans melanization with different catecholamine precursors.

Melanins are a class of natural pigments associated with a wide range of biological functions, including microbial virulence, energy transduction, and protection against solar radiation. Because of their insolubility and structural heterogeneity, solid-state nuclear magnetic resonance (NMR) spectroscopy provides an unprecedented means to define the molecular architecture of these enigmatic pigments. The requirement of obligatory catecholamines for melanization of the pathogenic fungus Cryptococcus neoformans also offers unique opportunities for investigating melanin development. In the current study, pigments produced with L-dopa, methyl-L-dopa, epinephrine, and norepinephrine precursors are compared structurally using (13)C and (1)H magic-angle spinning (MAS) NMR. Striking structural differences were observed for both aromatic and aliphatic molecular constituents of the mature fungal pigment assemblies, thus making it possible to redefine the molecular prerequisites for formation of the aromatic domains of insoluble indole-based biopolymers, to rationalize their distinctive physical characteristics, and to delineate the role of cellular constituents in assembly of the melanized macromolecules with polysaccharides and fatty acyl chain-containing moieties. By achieving an augmented understanding of the mechanisms of C. neoformans melanin biosynthesis and cellular assembly, such studies can guide future drug discovery efforts related to melanin-associated virulence, resistance to tumor therapy, and production of melanin mimetics under cell-free conditions.

Analogs of anthocyanins with a 3′,4′-dihydroxy substitution: Synthesis and investigation of their acid–base, hydration, metal binding and hydrogen-donating properties in aqueous solution

Abstract Glycosides of hydroxylated flavylium ions are proposed as pertinent analogs of anthocyanins, a major class of polyphenolic plant pigments. Anthocyanins with a 3′,4′-dihydroxy substitution on the B-ring (catechol nucleus) are especially important for their metal chelating and electron-donating (antioxidant) capacities. In this work, an efficient chemical synthesis of 3′,4′-dihydroxy-7- O - β - d -glucopyranosyloxyflavylium chloride and its aglycone is reported. Then, the ability of the two pigments to undergo proton transfer (formation of colored quinonoid bases) and add water (formation of a colorless chalcone) is investigated: at equilibrium the colored quinonoid bases (kinetic products) are present in very minor concentrations ( β - d -glucosyl moiety while the kinetics appears slower by a factor ca . 8. Although the glucopyranosyloxyflavylium ion and its aglycone display similar affinities for Al 3+ , the Al 3+ -glucoside complex is more stable than the Al 3+ -aglycone complex due to the higher sensitivity of the latter to water addition and conversion into the corresponding chalcone. Finally, the glucopyranosyloxyflavylium ion and its aglycone are compared for their ability to reduce the 1,1-diphenyl-2-picrylhydrazyl radical in a mildly acidic water/MeOH (1:1) mixture as a first evaluation of their antioxidant activity. Glycosidation at C 7 -OH results in a lower rate constant of first electron transfer to DPPH and a lower stoichiometry (total number of 1,1-diphenyl-2-picrylhydrazyl radicals reduced per pigment molecule). Anthocyanins are difficult to extract from plants in substantial amount. However, the analogs investigated in this work are of easy access by chemical synthesis and express the physico-chemical properties typical of anthocyanins. They can thus be regarded as valuable models for investigating the coloring, metal-binding and antioxidant properties of these important natural pigments.

Betalain production is possible in anthocyanin-producing plant species given the presence of DOPA-dioxygenase and L-DOPA

BackgroundCarotenoids and anthocyanins are the predominant non-chlorophyll pigments in plants. However, certain families within the order Caryophyllales produce another class of pigments, the betalains, instead of anthocyanins. The occurrence of betalains and anthocyanins is mutually exclusive. Betalains are divided into two classes, the betaxanthins and betacyanins, which produce yellow to orange or violet colours, respectively. In this article we show betalain production in species that normally produce anthocyanins, through a combination of genetic modification and substrate feeding.ResultsThe biolistic introduction of DNA constructs for transient overexpression of two different dihydroxyphenylalanine (DOPA) dioxygenases (DODs), and feeding of DOD substrate (L-DOPA), was sufficient to induce betalain production in cell cultures of Solanum tuberosum (potato) and petals of Antirrhinum majus. HPLC analysis showed both betaxanthins and betacyanins were produced. Multi-cell foci with yellow, orange and/or red colours occurred, with either a fungal DOD (from Amanita muscaria) or a plant DOD (from Portulaca grandiflora), and the yellow/orange foci showed green autofluorescence characteristic of betaxanthins. Stably transformed Arabidopsis thaliana (arabidopsis) lines containing 35S: AmDOD produced yellow colouration in flowers and orange-red colouration in seedlings when fed L-DOPA. These tissues also showed green autofluorescence. HPLC analysis of the transgenic seedlings fed L-DOPA confirmed betaxanthin production.ConclusionsThe fact that the introduction of DOD along with a supply of its substrate (L-DOPA) was sufficient to induce betacyanin production reveals the presence of a background enzyme, possibly a tyrosinase, that can convert L-DOPA to cyclo-DOPA (or dopaxanthin to betacyanin) in at least some anthocyanin-producing plants. The plants also demonstrate that betalains can accumulate in anthocyanin-producing species. Thus, introduction of a DOD and an enzyme capable of converting tyrosine to L-DOPA should be sufficient to confer both betaxanthin and betacyanin production to anthocyanin-producing species. The requirement for few novel biosynthetic steps may have assisted in the evolution of the betalain biosynthetic pathway in the Caryophyllales, and facilitated multiple origins of the pathway in this order and in fungi. The stably transformed 35S: AmDOD arabidopsis plants provide material to study, for the first time, the physiological effects of having both betalains and anthocyanins in the same plant tissues.

Anion sensitivity and spectral tuning of middle- and long-wavelength-sensitive (MWS/LWS) visual pigments.

The long-wavelength-sensitive (LWS) opsins form one of four classes of vertebrate cone visual pigment and exhibit peak spectral sensitivities (λ(max)) that generally range from 525 to 560 nm for rhodopsin/vitamin-A(1) photopigments. Unique amongst the opsin classes, many LWS pigments show anion sensitivity through the interaction of chloride ions with a histidine residue at site 197 (H197) to give a long-wavelength spectral shift in peak sensitivity. Although it has been shown that amino acid substitutions at five sites (180, 197, 277, 285 and 308) are useful in predicting the λ(max) values of the LWS pigment class, some species, such as the elephant shark and most marine mammals, express LWS opsins that possess λ(max) values that are not consistent with this 'five-site' rule, indicating that other interactions may be involved. This study has taken advantage of the natural mutation at the chloride-binding site in the mouse LWS pigment. Through the use of a number of mutant pigments generated by site-directed mutagenesis, a new model has been formulated that takes into account the role of charge and steric properties of the side chains of residues at sites 197 and 308 in the function of the chloride-binding site in determining the peak sensitivity of LWS photopigments.

The interplay of intermolecular interactions, packing motifs and electron transport properties in perylene diimide related materials: a theoretical perspective

Perylene diimide (PDI) and its derivatives hold great promise, since they are undeniably considered as an important family of organic n-type semiconductors with both high carrier mobilities and air stabilities comparable to p-type ones, although they traditionally stand out as a class of high-performance dyes and pigments. In this feature article, we summarize the influences of substituents on different positions (imide, ortho, bay) of PDI on their electronic and morphological (packing) properties, which are in close connection with the ability for carrier transport. Then representative molecular packing motifs for PDIs are also classified, with an emphasis on the intricate interplay of intermolecular interactions, packing motifs and electron transport properties of perylene imide related carrier transport materials from a theoretical point of view, towards paving the way for boosting and improving the electron transport mobilities and air stabilities of PDIs-based materials.

Marine sponge melanin: a new source of an old biopolymer

Various types of melanin have already been found in the majority of organisms, being this biopolymer considered as one of the major pigments present in nature. The presence of this pigment in marine sponges (Phylum Porifera, one of the simplest multicellular organisms) was postulated, but never characterized. In this context this work aims the extraction and characterization of a dark pigment observed in four different marine sponges species (Erylus mamillaris, Erylus discophorus var. deficiens, Pachymatisma johnstonia, Dercitus bucklandi). Characterization of the extracted biopolymer was performed using solid state analytical techniques, due to the characteristic non-solubility of melanin. Therefore, characterization techniques like SEM–EDS, IR, UV–vis, MALDI-MS, elemental analysis and X-ray powder diffraction (XRD) were used to identify the biopolymer. The results showed that the extracted material was obtained with high purity, being identified as melanin. The results also emphasize a large structure variability present in this pigment, showing different structure arrangements and composition depending on its source, which influences the UV behaviour. The structural characterization of this class of pigments is fundamental, allowing a better understanding of melanin properties.

Matrix assisted pulsed laser deposition of melanin thin films

Melanins constitute a very important class of organic pigments, recently emerging as a potential material for a new generation of bioinspired biocompatible electrically active devices. In this paper, we report about the deposition of synthetic melanin films starting from aqueous suspensions by matrix assisted pulsed laser evaporation (MAPLE). In particular, we demonstrate that it is possible to deposit melanin films by MAPLE even if melanin (a) is not soluble in water and (b) absorbs light from UV to IR. AFM images reveal that the film surface features are highly depending on the deposition parameters. UV-VIS and FTIR spectra show both the optical properties and the molecular structure typical of melanins are preserved.

Carotenoid and chlorophyll composition of commonly consumed leafy vegetables in Mediterranean countries

Major chloroplast pigments in five leafy vegetables (chicory-Cichorium intybus, cv. ‘Anivip’ and cv. ‘Monivip’, dandelion-Taraxacum officinale, garden rocket-Eruca sativa and wild rocket-Diplotaxis tenuifolia), commonly consumed in Mediterranean countries, have been separated by high-performance liquid chromatography (HPLC) on a reversed-phase column. Three classes of pigments were identified and quantified: xanthophylls (oxygenated carotenoids), carotenes (hydrocarbon carotenoids) and chlorophylls. The contents of the pigments in the analysed leafy vegetables varied significantly. The results indicated that selected leafy vegetables were moderately rich in xanthophylls, primarily lutein (3.87–7.44mg/100g fwt). Other xanthophylls were detected in relatively small quantities. The provitamin A carotenoids (α- and β-carotene) were also detected, but α-carotene were not present in chicory cultivars and in dandelion. The ratio of chlorophyll a/b varied from 2.44 to 2.67 depending on the species. The highest content of all the analysed constituents was found in the garden rocket.

Complex pigment evolution in the Caryophyllales

Carotenoids and flavonoids including anthocyanins are the predominant pigments in flowering plants, where they play important roles in pollination, seed dispersal, protection against stress and signalling. In certain families within the Pentapetalae order Caryophyllales, an unusual class of pigments, known as betalains, replaces the more common anthocyanins. This isolated occurrence of betalains in the Caryophyllales has stimulated over half a century of debate and experimentation. Numerous hypotheses have been suggested to explain the phylogenetically restricted occurrence of betalains and their apparent mutual exclusion with anthocyanins. In this review, we evaluate these hypotheses in the face of a changing interpretation of Caryophyllales phylogeny and new comparative genetic data. Phylogenetic analyses expose substantial gaps in our knowledge of the early evolution of pigments in the Caryophyllales and suggest pigmentation to be much more labile than previously recognized. Reconstructions of character evolution imply multiple switches from betalain to anthocyanin pigmentation, but also allow for possible multiple origins of betalains. Comparative genetic studies propose possible mechanisms underlying switches between pigment types and suggest that transcriptional down-regulation of late-acting enzymes is responsible for a loss of anthocyanins. Given these insights from molecular phylogenetics and comparative genetics, we discuss outstanding questions and define key goals for future research.

Combined non-destructive XRF and SR-XAS study of archaeological artefacts

We report on a non-destructive study of Sicilian ceramic fragments of cultural heritage interest, classified as "proto-majolica" pottery and dating back to the twelfth to thirteen centuries AD. The analytical approach used is based on the employment of two totally non-invasive spectroscopic techniques: X-ray fluorescence (XRF), using a portable energy-dispersive XRF analyser, and X-ray absorption spectroscopy, using synchrotron radiation as a probe (SR-XAS). XRF measurements allowed us to collect elemental and spatially resolved information on major and minor constituents of the decorated coating of archaeological pottery fragments, so providing preliminary results on the main components characterizing the surface. In particular, we assigned to Fe and Mn the role of key elements of the colouring agent. With the aim of obtaining more detailed information, we performed SR-XAS measurements at the Fe and Mn K-edges at the Italian BM08 beamline at the European Synchrotron Radiation Facility (Grenoble, France). The experimental data were analysed by applying principal component analysis and least-squares fitting to the near-edge part of the spectra (X-ray absorption near-edge structure) to determine the samples' speciation. From the overall results, umber, a class of brownish pigments characterized by a mixture of hydrated iron and manganese oxides, has been ascribed as a pigmenting agent.

Carotenoid Supplementation Positively Affects the Expression of a Non-Visual Sexual Signal

Carotenoids are a class of pigments which are widely used by animals for the expression of yellow-to-red colour signals, such as bill or plumage colour. Since they also have been shown to promote immunocompetence and to function as antioxidants, many studies have investigated a potential allocation trade-off with respect to carotenoid-based signals within the context of sexual selection. Although an effect of carotenoids on non-visual (e.g. acoustic) signals involved in sexual selection has been hypothesized, this has to date not been investigated. First, we examined a potential effect of dietary carotenoid supplementation on overall song rate during the non-breeding season in captive male European starlings (Sturnus vulgaris). After only 3–7 days, we found a significant (body-mass independent) positive effect of carotenoid availability on overall song rate. Secondly, as a number of studies suggest that carotenoids could affect the modulation of sexual signals by plasma levels of the steroid hormone testosterone (T), we used the same birds to subsequently investigate whether carotenoid availability affects the increase in (nestbox-oriented) song rate induced by experimentally elevated plasma T levels. Our results suggest that carotenoids may enhance the positive effect of elevated plasma T levels on nestbox-oriented song rate. Moreover, while non-supplemented starlings responded to T-implantation with an increase in both overall song rate and nestbox-oriented song, carotenoid-supplemented starlings instead shifted song production towards (reproductively relevant) nestbox-oriented song, without increasing overall song rate. Given that song rate is an acoustic signal rather than a visual signal, our findings therefore indicate that the role of carotenoids in (sexual) signalling need not be dependent on their function as pigments.