Sharks

Adult sharks were collected using longlines in the Raunefjord (Norway) between December 2009 and October 2012, transferred to Espeland Marine Station and housed in tanks placed in a dark cold room. Our protocol, including fish sacrifice, was in accordance with national guidelines for experimental fish care (fish handling approval #1664 was given by the National Committees for Research Ethics of Norway).

Photographs of spontaneous luminescence were taken in complete darkness with a Canon 7D camera (whole shark, sensitivity 6400 ISO, objective 20 mm; dorsal fins, 12800 ISO, 60 mm; both with an aperture of 2.8 and an exposure time of 30 s). For visibility purpose, a post adjustment of brightness and contrast was applied to the entire pictures using Adobe Photoshop®. Other pictures were taken with the same camera in natural light.

Luminescence recording

A Berthold FB12 luminometer coupled to an optical fibre was used to record in vivo intensity of dorsal and ventral continuous luminescence from several live specimens according to Claes et al.14. Values were corrected for fiber absorption and angular losses. Ventral photophore spacing/density and mean intensity of specimens was estimated according to Claes and Mallefet15, by counting the photophores present in standard (0.25 cm2) ventral skin patches under a binocular microscope.

Spine structure

Dorsal finspines were removed from dead specimens wrapped in plastic wrap with a small amount of fluid for hydration and packed tightly with paper towels into plastic tubes for micro-CT scanning (1174 scanner, SkyScan®, Kontich, Belgium). The X-ray source was set at 50kVp (peak kilovoltage) and 800 μA; 220 projections were acquired over an angular range of 360degrees. Samples were scanned with an isotropic voxel size of 16.8 μm, an integration time of 4500ms and a 0.25mm aluminum filter to decrease beam-hardening effects. Scans were reconstructed using commercial software (NRecon® SkyScan® software, version 1.6.1.2) then visualized in 3d and segmented virtually in multiple planes using Amira software (Mercury Computer Systems) to examine spine structure and morphology.

Histology

Skin tissues containing SAPs from dorsal fins of freshly dead specimens were excised and fixed in a 3.5% formaldehyde solution for two weeks. Tissues were progressively dehydrated (50, 70, 2 × 90%, 1 hour each), placed 1 h in 100% butanol and left overnight in 100% butanol at 60°C. Tissues were then submerged in paraffin wax for different periods (12 h, 1 h and 3 h) at melting temperature (58°C). Sections were performed with a metal knife microtome, stained using Masson's trichrome and photographed under a light/fluorescence microscope (Leitz Diaplan, Leitz, Wetzlar, Germany).

Spine transmission

The second dorsal finspines of freshly dead specimens were removed and their anterior-posterior spectral transmittance was measured near the tip, at 75% of the exterior spine length, using an Ocean Optics S2000 spectroradiometer. Broadband white light from an Ocean Optics PX-2 pulsed xenon lamp was delivered to the caudal surface of the spine via an optical fibre terminated with a quartz lens. Light transmitted anteriorly by the spine was collected via a second quartz lens coupled to an optical fibre connected to the spectroradiometer.

Visual modeling

The visibility ranges of both ventral and dorsal glows were calculated according to the theory developed by Nilsson et al.17. This range depends on the intensity of down-welling daylight and thus on depth in the sea. Etmopterus spinax was assumed to occur at ‘counterillumination depth’ where its silhouette, concealed by ventral photophores, is not detectable from below14. This depth was determined using the ventral photophore spacing (0.234 mm) and mean intensity (2.1 × 106 photons s−1) of the brightest shark in our dataset as inputs in the equation 7 in Supplemental Information from Nilsson et al.17. We adapted Nilsson et al. 's theory, originally developed for clear oceanic waters, by setting the beam attenuation and back-scattering coefficients to 0.3 m−1 and 0.0385 m−1, respectively, to agree with the turbid green waters of the fjords. The visibility range of SAP luminescence, modeled as a point source seen against a black background (the dorsal part of the shark's body), was then calculated at this counterillumination depth. The SAP luminescence intensity of the brightest shark (6.87 × 107 photons s−1) was used as input value in the equation 13 in Supplemental Information from Nilsson et al.17. This equation relates the maximum detection distance to the observer's pupil diameter. Possible observers include conspecifics, the shark's main prey (the pearlside fish Maurolicus muelleri)21 and potential predators such as piscivorous fishes (Galeus melastomus, Molva molva) and marine mammals (Phocoena phoecoena, Phoca vitulina). Pupil diameters of these animals were set to 7, 2, 12, 13, 8 and 14.4 mm, respectively, according to direct measurements (fishes) or published values (marine mammals34,35). Photoreceptor cell diameter, which plays a negligible role in the modeling17, was set to 3 μm.