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ESM 1 Michiels Et Al. Figure S1 Reflector Types Controlled ocular radiance in a diurnal fish looking at prey Nico K. Michiels, Victoria C. Seeburger, Nadine Kalb, Melissa G. Meadows, Nils Anthes, Amalia Mailli, Colin B. Jack ESM 1, figure S1 A. Retroreflector B. Specular reflector C. Diffuse reflector sends light back to source mirror-like reflection scatters light in all directions e.g. focusing eyes e.g. silvery structures e.g. matt white structures D. Reflective cup complex patterns of specular reflection e.g. ocelli with internal reflective layer Figure S1: Retroreflection in comparison to specular and diffuse reflection. 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. 1 Controlled ocular radiance in a diurnal fish looking at prey Nico K. Michiels, Victoria C. Seeburger, Nadine Kalb, Melissa G. Meadows, Nils Anthes, Amalia Mailli, Colin B. Jack ESM 2 List of all the species shown in figure 1. No. Species Marine/freshwater 1 Anomalops katoptron (Anomalopidae) Marine 2 Bryaninops natans (Gobiidae) Marine 3 Hyphessobrycon pulchripinnis (Characidae) Fresh 4 Serrasalmus nattereri (Characidae) Fresh 5 Barbonymus schwanenfeldii (Cyprinidae) Fresh 6 Leuciscus rutilus (Cyprinidae) Fresh 7 Ecsenius lividanalis (Blenniidae) Marine 8 Heteroconger hassi (Congridae) Marine 9 Ctenopharyngodon idella (Cryprinidae) Fresh 10 Danio malabaricus (Cyprinidae) Fresh 11 Spicara maena (Centracanthidae) Marine 12 Xyrichthys novacula (Labridae) Marine 13 Paracheirodon axelrodi (Characidae) Fresh 14 Pseudotropheus socolofi (Cichlidae) Fresh 15 Archamia macroptera (Apogonidae) Marine 16 Belontia signata (Belontiidae) Fresh 17 Epinephelus costae (Serranidae) Marine 18 Serranus cabrilla (Serranidae) Marine 19 Sarpa salpa (Sparidae) Marine 20 Anthias anthias (Serranidae) Marine 21 Labrus mixtus (Labridae) Marine 22 Caesio cuning (Caesionidae) Marine 23 Sebastes caurinus (Sebastidae) Marine 24 Embiotoca lateralis (Embiotocidae) Marine 1 Controlled ocular radiance in a diurnal fish looking at prey Nico K. Michiels, Victoria C. Seeburger, Nadine Kalb, Melissa G. Meadows, Nils Anthes, Amalia Mailli, Colin B. Jack ESM 3, figure S2, A, B, C and D Experimental setup and spectral properties of ocular sparks Figure S2-A: Frontal view of three of the 21 experimental tanks in the setup, each section with its own blue LED source. A Tripterygion delaisi 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). Figure S2-B: Side view of the experimental setup (not drawn to scale) including a copepod chamber (front view shown at the top). Figure S2-C: Log10-transformed mean photon radiance as a function of wavelength for red ocular sparks (solid red line), other parts of the red fluorescent iris (dashed red line), blue ocular sparks and a diffuse white reflectance standard (blue dotted line) under blue LED illumination in the experimental room. The insert (top right) shows the intensity of the red ocular spark relative to the regular fluorescent iris emission (thin black line), showing that it is up to 6 times more intense. The vertical dashed line at 530 nm separates the blue LED excitation range and associated reflections (left) from the fluorescent emissions (right). Measurements taken with a calibrated radiospectrometer (PhotoResearch SpectraScan PR 740 or PR 670) and represent the mean of the strongest measured from five individuals each for red and blue ocular sparks. 3.5 3.0 2.5 2.0 1.5 1.0 Blue ocular spark reflectance 0.5 0.0 400 450 500 550 600 650 700 Wavelength (nm) Figure S2-D: Reflectance (proportion) of the blue ocular spark in live fish in the visual spectrum relative to a white diffuse reflectance standard (dashed line) measured under broad- spectrum, downwelling illumination. Measurements taken each time the fish was showing a blue ocular spark (multiple measurements per fish). Thin lines: maximum reflectance spectra obtained from each of 5 different individuals. Thick line: overall average. Measurements taken with a PhotoResearch SpectraScan PR 670 radiospectrometer (with optics) aimed into a saltwater-filled tank with the fish and a Spectralon diffuse white standard for comparison. Controlled ocular radiance in a diurnal fish looking at prey Nico K. Michiels, Victoria Seeburger, Nadine Kalb, Melissa G. Meadows, Nils Anthes, Amalia Mailli, Colin Bruce Jack ESM 6, figure S3 2.0 Strongest$ocellus$ 2.0 Strongest$ocellus$ max$ 2.0 Strongest$ocellus,$$ 1.8 1.8 1.8 +SD$ rel.%to%specular%mirror% 1.6 1.6 1.6 1.4 1.4 1.4 mean$ 1.2 1.2 1.2 1.0 1.0 Y 1.0 0.8 0.8 *SD$ 0.8 0.6 0.6 0.6 Ocellus reflectance 0.4 Ocellus reflectance 0.4 min$ 0.4 0.2 0.2 0.2 0.0 0.0 0.0 400 450 500 550 600 650 700 400 450 500 550 600 650 700 400 450 500 550 600 650 700 Wavelength (nm) Wavelength in 10 nm classes 1.0 2.0 Intermediate$ocellus$ 2.0 Weakest$ocellus$ Central$area$of$eye$ 1.8 1.8 0.8 1.6 1.6 0.6 0.4 Reflectance$(prop.)$ 1.4 1.4 1.2 1.2 0.2 1.0 1.0 1.00.0 Cephalothorax$ 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0.0 0.0 0.0 400 450 500 550 600 650 700 400 450 500 550 600 650 700 400 450 500 550 600 650 700 Wavelength (nm) Wavelength (nm) Wavelength$(nm)$ Figure S4: Spectral reflectance of all three ocelli of 22 live Tigriopus californicus copepods expressed as a proportion of the coaxial light reflected relative to a Spectralon diffuse reflectance standard (or a metal mirror top right). Top row shows the measurements of the brightest of three ocelli only, showing all curves (top left, n = 22 copepods), a statistical summary (top centre) and reflectance relative to a metal mirror (top right). Bottom: statistical summaries for the ocellus of intermediate (left) and lowest brightness (centre), as well as for the central red pigmented area of the eye and the cephalothorax (cuticle). Variation in ocellus brightness does not depend on ocellus identity, but on orientation of that ocellus relative to the observer. Methods: Live copepods were glued to the tip of a fine insect pin using Surgibond superglue and submerged in a seawater-filled diffuse white Teflon dish with the nauplius eye facing upward. The sample was observed under a Leica DM 5000B microscope using a Leica HCX APO 40x/0.80 U-V-I lens for liquid cell cultures. The microscope was modified to allow for perfect coaxial illumination using a cold light source (Schott KL 2500). Radiance measurements were taken with a PhotoResearch PR-740 radiospectrometer through the microscope. Eyeshine measurements were repeated 2-3 times per ocellus. Internal glare in the microscope accounted for less than 1% of the signal and was subsequently ignored. The graphs show that in this setup, copepod ocelli reflect up to two times as strong as a diffuse white standard and up to 50% as strong as a specular mirror. The high value of the diffuse white standard relative to the mirror can be attributed by the short (3.3 mm) working distance of the objective, allowing much of the diffusely reflected light to be captured by the nearby lens. .
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