From opsin gene expression to visual performance: an investigation into the multiple spectral sensitivities of boxfishes (Ostracion spp.).

Colour vision arises from the comparison of spectral input across at least two differently tuned photoreceptors. In most vertebrates, colour discrimination is mediated by two to four differently tuned cone photoreceptors, providing sensitivity from ultraviolet (<400 nm) to red (∼600-700 nm) wavelengths. However, microspectrophotometry (MSP) indicated that the white-spotted boxfish, Ostracion meleagris, collected from Hawaiian reefs could possess five spectrally distinct cones. To explore the potential for increased cone diversity, we used a multidisciplinary approach to investigate colour vision in O. meleagris and its sister species, Ostracion cubicus, from the Great Barrier Reef. Contrary to the MSP data, retinal transcriptomics and fluorescence in situ hybridisation found no evidence for five spectral sensitivities in either species. Instead, single cones (PR3) expressed sws2b, while the two members of the double cones (PR1 and PR2) expressed rh2a and rh2c opsins, respectively. Amino acid modelling revealed putative visual pigments with uniformly spaced peak spectral sensitivities at 420 nm (SWS2B-based), 461-474 nm (RH2C) and 523-528 nm (RH2A). We compared the colour vision performance of O. cubicus with that of a well-studied trichromatic reef fish, the Picasso triggerfish (Rhinecanthus aculeatus). Compared with R. aculeatus, O. cubicus exhibited significantly lower detection thresholds for blue and achromatic grey, but not for green stimuli. Together, we found no support for five distinct spectral channels in the colour vision system in Australian boxfishes. Instead, these boxfish likely have trichromatic colour vision optimised for contrast detection, highlighting the importance of integrating multiple lines of investigation for accurately characterising animal colour perception.