Red fluorescence in reef fish: a novel signalling mechanism?

Abstract

Background: At depths below 10 m, reefs are dominated by blue-green light because seawater selectively absorbs the longer, 'red' wavelengths beyond 600 nm from the downwelling sunlight. Consequently, the visual pigments of many reef fish are matched to shorter wavelengths, which are transmitted better by water. Combining the typically poor long-wavelength sensitivity of fish eyes with the presumed lack of ambient red light, red light is currently considered irrelevant for reef fish. However, previous studies ignore the fact that several marine organisms, including deep sea fish, produce their own red luminescence and are capable of seeing it.

Results: We here report that at least 32 reef fishes from 16 genera and 5 families show pronounced red fluorescence under natural, daytime conditions at depths where downwelling red light is virtually absent. Fluorescence was confirmed by extensive spectrometry in the laboratory. In most cases peak emission was around 600 nm and fluorescence was associated with guanine crystals, which thus far were known for their light reflecting properties only. Our data indicate that red fluorescence may function in a context of intraspecific communication. Fluorescence patterns were typically associated with the eyes or the head, varying substantially even between species of the same genus. Moreover red fluorescence was particularly strong in fins that are involved in intraspecific signalling. Finally, microspectrometry in one fluorescent goby, Eviota pellucida, showed a long-wave sensitivity that overlapped with its own red fluorescence, indicating that this species is capable of seeing its own fluorescence.

Conclusion: We show that red fluorescence is widespread among marine fishes. Many features indicate that it is used as a private communication mechanism in small, benthic, pair- or group-living fishes. Many of these species show quite cryptic colouration in other parts of the visible spectrum. High inter-specific variation in red fluorescence and its association with structures used in intra-specific signalling further corroborate this view. Our findings challenge the notion that red light is of no importance to marine fish, calling for a reassessment of its role in fish visual ecology in subsurface marine environments.

Figure 1

General introduction to light attenuation and observation of natural fluorescence in near-shore marine environments. a. The visual spectrum ranges from 400 to 700 nm at the water surface, but downwelling sunlight loses the red component (600–700 nm) rapidly within 10–15 m (modified from Pinet PR (2000) Invitation to Oceanography. Jones and Bartlett). UV and violet wavelengths are attenuated less rapidly. The attenuation with depth of spectral composition (and light intensity, not shown) varies strongly with the concentration of organic matter in the water column. b. Most red pigmentation is based on reflectance of the red component of ambient light and therefore only appears "red" when close to the surface during daytime or under broad spectral light (e.g. dive torch). Fish with this pigmentation appear dull grey in deeper water. Red fluorescent patterns, however, continue to appear reddish and bright, even in deeper water, where excitation of fluorescent pigments by shorter wavelengths induces redness. Note that red fluorescence is rarely perceived as pure red, but is mostly an enhancer of mixed colours such as pink, lilac or red brown. Even so, it remains clearly visible in deeper water as a contrast enhancer. Closer to the surface, fluorescent patterns are masked by reflective colouration (e.g. yellow and red in Eviota pellucida, Fig. 3). c. Since excitation frequencies (blue-green) are brighter than emission frequencies (red in our example) red fluorescence is best seen when viewed through a filter that blocks the excitation frequencies and only allows the emission frequencies to pass. When looking through a red filter in e.g. 20 m depth, all remaining red light must be "locally produced" through fluorescence or bioluminescence. Given that fluorescence exploits light energy from ambient light, it is more efficient than bioluminescence and therefore likely to be the mechanism of choice for diurnal fish.

Figure 2

Examples of common red fluorescent invertebrates on coral reefs. a-d. Stony corals (a. Goniopora, b. Mycedium, c. Fungia, d. Porites). e. Reef scenery, as seen through a Lee Medium Red filter. f. Unidentified alga (white pingpong ball as reflectance reference). g. Calcareous alga Amphiroa. h. Polychaete worm Sabellastarte indica. i. Typical environment of S. indica under reef ledge. Pictures a-d and f-h show object under natural illumination (left) and as seen through a red filter (right). Pictures e and i show fluorescence in the field as seen by a digital camera. Most pictures taken in the field (Dahab, Egypt) under natural illumination between 14 and 17 m depth. Only c was photographed in the laboratory. Other reef invertebrates seen to fluoresce were sponges (e.g. Aaptos, Acanthella, Theonella) and feather stars (e.g. Colobometra, Oligometra).

Figure 3

How to distinguish a red fish from a red fluorescent fish?. Comparison between a non-fluorescent goby, Trimma cana (left), and a similar sized, red fluorescent goby, Eviota pellucida (right) under four viewing conditions. a. Artificial white light from a strong Schott KL 2500 LCD halogen cold light source under a Leica stereomicroscope (MZ 16F). b. In a halogen-illuminated aquarium with downwelling light filtered through Lee 729 Scuba-Blue filter (transmission range 400–550 nm, λ_max = 500 nm), thus simulating light at depth. c. Illumination as in b, but viewed through a red filter, revealing red fluorescence. d. Illumination as in a, seen under a Leica fluorescence stereomicroscope (MZ 16F) using green light for excitation, while viewing through red filter. The differences between the viewing conditions illustrate that red fluorescence can only be reliably seen when excitation and emission frequencies are separated, as at depth in the sea or under blue light, and by using a red filter for viewing.

Figure 4

Red fluorescent representatives of five different reef fish families. a. Eviota pellucida (Gobiidae). b. Pseudocheilinus evanidus (Labridae). c. Corythoichthys flavofasciatus and d. C. schultzi (Syngnathidae), e. Enneapterygius pusillus, f. E. destai, g. E. abeli and h. Helcogramma steinitzi (Tripterygiidae). i. Ecsenius dentex and j. Crossosalarias macrospilus (Blenniidae). All pictures are from the laboratory, except for j (field). Left: broad spectrum illumination, right: red fluorescence under blue (laboratory) or natural (field) illumination.

Figure 5

Diversity in red fluorescence in 14 goby species. a. Bryaninops natans. b. B. yongei. c. Ctenogobiops tangaroai. d. Gnatholepis anjerensis. e. Istigobius decoratus. f. Fusigobius duospilus. g. F. longispinus. h. Pleurosicya micheli. i. P. prognatha. j. Eviota guttata. k. E. prasina. l. E. zebrina. m. E. sebreei. n. Trimma avidori. All fish shown under broad spectrum illumination (left) in the laboratory (halogen) or field (a, b and h) and under blue illumination (right) with red filter.

Figure 6

Spectrometric measurements confirm the emission of red light, and the ability to see it. a. Fluorescence emission spectra of five genera of Gobiidae (top), one genus of Syngnathidae, Labridae and Blenniidae each (middle) and three species of Tripterygiidae (bottom). b. Absorptance spectra of photoreceptor visual pigments found in Eviota pellucida with wavelengths of maximum absorptance (λmax) at 497 (rods), 458 (SWS single cones), 528 (MWS, twin cones) and 540 nm (LWS, twin cones). The fluorescence emission spectrum of E. pellucida is included for comparison (dashed line).

Figure 7

Sources of red fluorescence in fishes. a. Guanine crystals from the eye ring of Eviota pellucida (fluorescence microscopy). b. Same from Ctenogobiops tangaroai (fluorescence overlaid with phase contrast). c. As in b, diamond form (fluorescence microscopy). d. Guanine crystals falling apart in characteristic platelets (from eye ring of E. pellucida, scanning electron micrograph). e. Single red fluorescent guanine crystal among normal crystals in eye ring of the non-fluorescent goby Trimma cana (fluorescence microscopy, see also Fig. 3). f. Scale of Pseudocheilinus evanidus, in which the red fluorescent pigment is associated with bony scales and fin rays (fluorescence microscopy). Scale bar = 50 μm unless indicated otherwise.

Figure 8

Red fluorescence in a signalling context. a-b. Frameshots of two videos suggestive of communication involving fluorescent fins. a. Enneapterygius destai waving its first dorsal fin when 'excited' (laboratory, from Additional file 3, sequence 2b). b. Corythoichthys schultzi pair interacting, displaying conspicuous red fluorescence on the tail plate (natural reef illumination, 20 m depth, from Additional file 3, sequence 1). c. Ctenogobiops maculosus is cryptic when sitting at its burrow entrance under natural illumination (insert), but shows conspicuous eyes when emphasizing red wavelengths (Additional file 2). d. Bryaninops natans with a pupil-like black spot on the upper part of the fluorescent eye ring suggesting that fluorescence and (deceptive) gaze signalling may be linked in this species (Additional file 2).