A complex carotenoid palette tunes avian colour vision

Abstract

The brilliantly coloured cone oil droplets of the avian retina function as long-pass cut-off filters that tune the spectral sensitivity of the photoreceptors and are hypothesized to enhance colour discrimination and improve colour constancy. Although it has long been known that these droplets are pigmented with carotenoids, their precise composition has remained uncertain owing to the technical challenges of measuring these very small, dense and highly refractile optical organelles. In this study, we integrated results from high-performance liquid chromatography, hyperspectral microscopy and microspectrophotometry to obtain a comprehensive understanding of oil droplet carotenoid pigmentation in the chicken (Gallus gallus). We find that each of the four carotenoid-containing droplet types consists of a complex mixture of carotenoids, with a single predominant carotenoid determining the wavelength of the spectral filtering cut-off. Consistent with previous reports, we find that the predominant carotenoid type in the oil droplets of long-wavelength-sensitive, medium-wavelength-sensitive and short-wavelength-sensitive type 2 cones are astaxanthin, zeaxanthin and galloxanthin, respectively. In addition, the oil droplet of the principal member of the double cone contains a mixture of galloxanthin and two hydroxycarotenoids (lutein and zeaxanthin). Short-wavelength-absorbing apocarotenoids are present in all of the droplet types, providing filtering of light in a region of the spectrum where filtering by hydroxy- and ketocarotenoids may be incomplete. Thus, birds rely on a complex palette of carotenoid pigments within their cone oil droplets to achieve finely tuned spectral filtering.

Keywords: carotenoid; hyperspectral microscopy; microspectrophotometry; vision.

Figure 1.

(a) Brightfield image of a flat-mounted P21 chicken retina showing the distinctive coloration of the cone oil droplets (scale bar = 10 µm). (b) A schematic representation of the photoreceptor subtypes of the avian retina: rods, short-wavelength sensitive type 1 (SWS1), short-wavelength sensitive type 2 (SWS2), medium-wavelength sensitive (MWS), long-wavelength sensitive (LWS), and double cones. The oil droplet is coloured to approximate its appearance in the brightfield image. Figure modified from [6]. (c–f) Normalized absorbance spectra of the major (c,d) apocarotenoids, (e) hydroxycarotenoids and (f) ketocarotenoids found in the avian retina. The molecular structures of representative carotenoids are shown in (g) with the double bonds in conjugation (highlighted in red) that contribute to the absorbance spectra.

Figure 2.

(a) False colour composite image of the cone oil droplets showing the relative contributions of the four spectral components to the droplet emission spectra, as measured by hyperspectral fluorescence microscopy and defined by MCR analysis. (b–e) The multivariate curve resolution (MCR) spectral components derived from the cone oil droplet emission spectra overlaid with the best matched emission spectra of the pure preparations of the major carotenoids in the avian retina. Right: detailed view of the major resonance peak. (f) The peak emission intensity per microgram of the pure carotenoid preparations.

Figure 3.

(a) Oil droplet groupings identified by the PAM clustering analysis of the relative intensity of the four MCR components of the emission spectra. The x- and y-axes are principal components (PC) 1 and 2 derived from the MCR spectral components. The contributions of these components are represented by the arrows superimposed on the plot. (b) A stacked bar chart shows the mean relative intensity of the four MCR components of the emission spectra within each of the droplet groups.

Figure 4.

(a) An unexpanded R-type droplet imaged through the MSP system. The measurement beam is located to the right of the droplet. (b) The mean normalized absorbance spectra of the unexpanded oil droplets measured with the beam MSP. The standard error of the mean is shown as the grey interval around each line. The apparent transmission at short wavelengths is an artefact of light scattering around the optically dense droplets. (c) The same droplet shown in (a) after expansion with mineral oil. (d) The mean ± s.e. normalized absorbance spectra of the oil droplets after expansion with mineral oil.

Figure 5.

The pure (left) or mixed (middle) carotenoid spectra that best fit the spectra of expanded (a) C-type, (b) P-type, (c) Y-type and (d) R-type oil droplets. The mean ± s.e. proportion of each carotenoid type in the mixed carotenoid fit (middle) is shown in the bar graphs to the right of each curve.

Figure 6.

An example of the absorptance spectra over a range of optical densities calculated from (a) the expanded Y-type droplet spectrum and (b) the pure zeaxanthin spectrum. These calculated spectra were used to predict λ_cut and short-wavelength transmittance of the pure carotenoids over a range of optical densities. (c) The measured mean ± s.e. of λ_cut and optical density (OD) of the cone oil droplets (points) overlaid on the λ_cut predicted for pure carotenoid spectra over a range of optical densities. (d) The measured mean ± s.e. short-wavelength (350–400 nm) transmittance and OD of the cone oil droplets (points) overlaid on the short-wavelength transmittance predicted for pure carotenoid spectra over a range of optical densities.