Analytical Volume 6 Number 5 7 March 2014 Pages 1263–1610 Methods
www.rsc.org/methods
ISSN 1759-9660
PAPER Daniel B. Thomas et al. Non-destructive descriptions of carotenoids in feathers using Raman spectroscopy Analytical Methods
PAPER
Non-destructive descriptions of carotenoids in feathers using Raman spectroscopy† Cite this: Anal. Methods,2014,6,1301 Daniel B. Thomas,*ab Kevin J. McGraw,c Helen F. Jamesa and Odile Maddenb
Chemical analyses of pigments in skin, scales, feathers and fur have provided deep insight into the colouration and visual communication strategies of animals. Carotenoid pigments in particular can be important colour signals in birds and other animals. Chromatographic analyses of plumage carotenoids require the destruction of one or more feathers, which has made pigment research on threatened species or museum specimens challenging. Here we show that Raman spectroscopy, coupled with multivariate statistics, can be used to identify the most abundant carotenoid within a single feather barb without sample destruction. Raman spectra from the feathers of 36 avian species were compared to data on pigment presence from high-performance liquid chromatography. Feathers rich with a- doradexanthin, astaxanthin, canary xanthophylls, canthaxanthin, cotingin or lutein were discriminated by subtle shifts in Raman spectral band positions, and by novel bands associated with particular carotenoids. As an example application of this method, we predicted the most abundant carotenoid in the plumage Received 24th October 2013 of selected Australian and New Zealand songbirds. a-Doradexanthin is predicted in the plumage of Accepted 2nd December 2013 Petroica robins from Australia, whereas Petroica immigrants to New Zealand display a yellow carotenoid DOI: 10.1039/c3ay41870g that is likely lutein. Raman spectroscopy is useful for non-destructive studies of carotenoids and is well- www.rsc.org/methods suited for analysing large ornithological museum collections.
1 Introduction many bird feathers are pigmented with carotenoids and can be important for mate choice.6 Several types of carotenoid occur in Many animals use pigments in the integument (i.e. in skin, the dietary items of birds, including yellow lutein in many plants scales, feathers or fur) for camouage or visual communication. and red astaxanthin in several invertebrates.7,8 Some birds Classic examples of pigments as visual signals include the display dietary carotenoids in their plumages (e.g. European black, red and yellow warning stripes across the scales of a coral greennch Chloris chloris, American amingo Phoenicopterus snake (Micrurus fulvius) and the orange and ultraviolet-reect- ruber), whereas other species deposit new carotenoids modied ing scales of sulphur butteries (Colias eurytheme) that advertise from dietary pigments into plumage (e.g. Atlantic canary, Serinus – individual quality.1,2 From chemical analyses of integumentary canaria).8 10 Modied carotenoids and their dietary precursors pigments, we have gained deep insight into both how and why can produce substantially different plumage colours (e.g. lutein animals communicate in colour.3 vs. cotingin in pompadour cotinga, Xipholena punicea).11 Red, orange, and yellow carotenoid pigments are abundant Researchers previously have used mass spectrometry and high- in organisms, ranging from plants, where they serve accessory performance liquid chromatography (HPLC) to identify at least photosynthetic roles, to animals, where they can play key roles 25 carotenoid compounds in feathers from ca. 200 bird species.5 in sexual advertisement. Studies of carotenoids in animals have Typically, studies assessing bird plumage pigments have provided important insights for elds as diverse as evolutionary relied on destructive sampling of tissue to remove the pigment biochemistry, nutritional ecology and sexual selection.4,5 from the feather matrix for subsequent chemical analysis (i.e. Regarding sexual selection, the red, orange and yellow hues of HPLC). This sampling scheme limits our ability to either study threatened species or to make good use of specimens in museum collections, where large-scale tissue collection is aDepartment of Vertebrate Zoology, National Museum of Natural History, Smithsonian discouraged. We sought to test a non-destructive technique, Institution, Washington, DC, 20013, USA. E-mail: [email protected]; Fax: Raman spectroscopy, for identifying the most abundant carot- +1 202 633 8084; Tel: +1 202 425 7270 bMuseum Conservation Institute, Smithsonian Institution, Suitland, MD, 20746, USA enoid pigment in bird feathers. We aimed to determine if cSchool of Life Sciences, Arizona State University, Tempe, AZ, 85287, USA Raman spectroscopy could provide pigment information that † Electronic supplementary information (ESI) available: Specimens analysed in may be relevant to evolution, physiology or behavioural studies the current study (Table S1); description of ancestral state reconstruction for of birds. Modern Raman spectroscopy is used to study the Petroicidae; Petroicidae phylogeny with plumage characters coded (Fig. S1). See energy exchanged between laser photons and a target sample, DOI: 10.1039/c3ay41870g
This journal is © The Royal Society of Chemistry 2014 Anal. Methods,2014,6,1301–1308 | 1301 Analytical Methods Paper which provides information about covalent bonds and thus barb. Spectra were measured using a Nicolet Almega XR spec- about the molecules or minerals in a sample. The mechanism trometer (Thermo Electron Corporation, Madison, WI, USA), underpinning the brilliant colouration of a carotenoid housed at the Museum Conservation Institute, Smithsonian compound is also responsible for producing a vivid Raman Institution. Feathers did not undergo any specialised sample spectrum; all carotenoids have a conjugated backbone, and treatment and were placed on a microscope stage for analysis. variations in conjugation length, terminal cyclisation and Feathers were probed with a 780 nm 150 mW diode laser, functional groups distinguish different carotenoids,5 because through a 50 Mplan apochromatic objective lens (Olympus, they inuence the wavelengths of light absorbed by carotenoids Melville, NY, USA) and 100 mm pinhole aperture (BX51 confocal and the strength of vibrations between atoms. In previous microscope, Olympus, Melville, NY, USA). Carotenoids have a studies, both Veronelli et al.12 and Withnall et al.13 related shis very broad pre-resonance range,16,17 and hence the spectra in Raman peak positions to the lengths of conjugated back- collected with the 780 nm excitation wavelength were analyti- bones, and more recently, Jehliˇcka et al.14 used Raman peak cally useful, and were comparable to spectra collected with 532 positions to distinguish the different carotenoid compositions nm excitation (532 nm spectra not shown). The green wave- of microbial cultures. Hence, Raman spectra might be used to length may be more sensitive to uorescent impurities (i.e. co- identify specic carotenoids. deposited melanin) and thus our study used the less sensitive Raman spectra of carotenoids in feathers were rst docu- near infrared wavelength. Future studies may wish to evaluate mented by Veronelli et al.,12 and more recently Mendes-Pinto the benets of using specic excitation wavelengths for partic- et al.15 showed the inuence of binding proteins on both the ular feathers. Scattered light was collected with a Peltier-cooled light-absorption properties and Raman spectra of feather CCD detector and each spectrum was a co-addition of 32 scans carotenoids in a species of purpletu (genus Iodopleura). Both across 100–3500 cm 1 (2.6–4.9 cm 1 spectral resolution). A of these earlier studies affirmed that Raman spectra of feathers spectrum of a polystyrene standard was collected at the begin- vary with carotenoi