Spectra of excitation and emission of the fluorescent pigment in fluorescent chromatophores.

<p>Fluorescence has a maximum at around 595 nm (dashed line) with an optimal excitation wavelength around 500 nm (solid line).</p

Morphology of a fluorescent chromatophore.

<p>Confocal Laser scanning microscopy, excitation 510 nm. Fluorosomes are the only visible structures within the fluorescent cell, but in the dispersed state they show the cell outline as they fill the cytoplasm entirely. Scale bar  = 30 µm.</p

Distribution of erythrophores, melanophores and fluorescent chromatophores in the interradial membrane of a dorsal fin of <i>E. pellucida</i>.

<p><b>a</b>) Erythrophores (red) and melanophores (black) are visible in bright field microscopy. <b>b</b>) Fluorescent chromatophores appear in fluorescence microscopy. <b>c</b>) Overlay of a) and b). Note that erythrophores, melanophores and fluorescent chromatophores are spatially distributed and can be distinguished. Scale bar  = 400 µm.</p

Results of cell manipulation.

<p>Bars show results of neuronal and hormonal manipulation in terms of normalized fluorescent area in interradial membranes of <i>E. pellucida</i> before (white bars) and after treatment (shaded bars, respectively). Neuronal K⁺ stimulation significantly decreased fluorescent area (paired t-test, t = 10.5, df = 5, p<0.005). The Lidocain-treatment effectively inhibited this effect (paired t-test, t = 1.86, df = 4, p>0.13). Neurotransmitter-induced aggregation of fluorosomes (NA) was highly significant (paired t-test, t = 4.67, df = 9, p<0.001). Aggregation induced by MCH was significant (paired t-test, t = 5.19, df = 3, p<0.013) as well as α–MSH significantly induced dispersal in pre-aggregated cells (paired Wilcoxon, Z = 10.5, df = 5, p = 0.03). Bars include standard errors.</p

Aggregation of fluorescence.

<p>Classified according to melanophore index 5 (a) through melanophore index 1 (e, respectively). In a completely dispersed state (a) the nucleus becomes visible as there is only little cytoplasm one the apical and basal side of the nucleus in these flat cells. In the aggregated state (e), the nucleus is tightly packed with fluorosomes. Scale bar  = 100 µm.</p

Figure S1 The mean transmittance of the dermal cornea, scleral cornea, and lens of the triplefin Tripterygion delaisi from Red fluorescence of the triplefin <i>Tripterygion delaisi</i> is increasingly visible against background light with increasing depth

The light environment in water bodies changes with depth due to the absorption of short and long wavelengths. Below a 10 m depth, red wavelengths are almost completely absent, rendering any red-reflecting animal dark and achromatic. However, fluorescence may produce red coloration even when red light is not available for reflection. A large number of marine taxa including over 270 fish species are known to produce red fluorescence, yet it is unclear under which natural light environment fluorescence contributes perceptively to their colours. To address this question we: (i) characterized the visual system of <i>Tripterygion delaisi,</i> which possesses fluorescent irides, (ii) separated the colour of the irides into its reflectance and fluorescence components and (iii) combined these data with field measurements of the ambient light environment to calculate depth-dependent perceptual chromatic and achromatic contrasts using visual modelling. We found that triplefins have cones with at least three different spectral sensitivities, including differences between the two members of the double cones, giving them the potential for trichromatic colour vision. We also show that fluorescence contributes increasingly to the radiance of the irides with increasing depth. Our results support the potential functionality of red fluorescence, including communicative roles such as species and sex identity, and non-communicative roles such as camouflage

<i>Tripterygion delaisi</i> showing ocular sparks in the field (video) from Controlled iris radiance in a diurnal fish looking at prey

Free-roaming <i>T. delaisi</i> individuals filmed while foraging on natural substrates in 5 m depth in Stareso, Corsica. Individuals show blue and red ocular sparks frequently and switch between them, or turn them off, by small eye movements. Ocular sparks require the fish to sit in a sun-lit environment and are generated by focusing downwelling light on the iris or skin below the pupil. See also figure 2 in the main text

Comparison between four common reflector types (figure) from Controlled iris radiance in a diurnal fish looking at prey

Comparison between four common reflector types. A. Focusing eyes work as a retroreflector. By focusing light from an object onto the back of the eye structure, the reflected light is sent back to the source. The brightness of the returned light depends on the reflectiveness of the layer behind the lens (e.g. a tapetum). B. Specular mirrors reflect the light at an angle that is identical to the incoming angle. They send light back to the source only when it arrives orthogonal to the mirror's surface. Silvery fish scales have specular properties. C. Diffuse reflectors scatter incoming light in all directions. Matt structures are diffuse reflectors. D. Reflective cups show complex patterns of specular reflection, but have a higher probability to send light back to the source than a flat specular mirror. Although this may explain the strength and directionality of the reflections seen in the eyes of some invertebrates such as copepods, the actual reflective properties of copepod eyes remain to be investigated

Experimental design (2 figures, A and B) from Controlled iris radiance in a diurnal fish looking at prey

<b>Figure S5-A:</b> Frontal view of three of the 21 experimental tanks in the setup, each section with its own blue LED source. A <i>Tripterygion delaisi</i> individual is visible at the front window of the middle tank. The top image shows the section with manual white balance, which approximates how humans perceive the setup once adapted to the blue light. Setting the camera to automatic white balance (below) illustrates how the setup appears to a human immediately upon entering the room from a regular, broad-spectral environment. We do not know how fish perceive colour in a blue environment like this – but a certain degree of colour constancy (neural compensation for a skewed ambient spectrum), as in the top image, is expected (images taken with a Nikon AW130 by Gregor Schulte).<b>Figure S5-B:</b> Side view of the experimental setup (not drawn to scale) including a copepod chamber (front view shown at the top)

Live copepods (<i>Tigriopus californicus</i>) under diffuse, coaxial illumination (video) from Controlled iris radiance in a diurnal fish looking at prey

The recording demonstrates the reflectiveness of copepod ocelli. As can be seen, the reflected beam is not consistently sent back to the source, but sometimes also in other directions. Although this is not true retroreflection, the directionality and intensity of the reflection confirms a form of specular reflection, as expected for the guanine crystals that cover the inside of <i>T. californicus</i> cup-like ocelli [1] (see also ESM S1) (video by N.K.M.).1. Martin, G. G., Speekmann, C., Beidler, S. 2000 Photobehavior of the harpacticoid copepod Tigriopus californicus and the fine structure of its nauplius eye. Invertebr Biol. 119, 110-124. (10.1111/j.1744-7410.2000.tb00179.x