Behavioral photosensitivity of multi-color-blind medaka: enhanced response under ultraviolet light in the absence of short-wavelength-sensitive opsins.

BACKGROUND: The behavioral photosensitivity of animals could be quantified via the optomotor response (OMR), for example, and the luminous efficiency function (the range of visible light) should largely rely on the repertoire and expression of light-absorbing proteins in the retina, i.e., the opsins. In fact, the OMR under red light was suppressed in medaka lacking the red (long-wavelength sensitive [LWS]) opsin. RESULTS: We investigated the ultraviolet (UV)- or blue-light sensitivity of medaka lacking the violet (short-wavelength sensitive 1 [SWS1]) and blue (SWS2) opsins. The sws1/sws2 double or sws1/sws2/lws triple mutants were as viable as the wild type. The remaining green (rhodopsin 2 [RH2]) or red opsins were not upregulated. Interestingly, the OMR of the double or triple mutants was equivalent or even increased under UV or blue light (λ = 350, 365, or 450 nm), which demonstrated that the rotating stripes (i.e., changes in luminance) could fully be recognized under UV light using RH2 alone. The OMR test using dichromatic stripes projected onto an RGB display consistently showed that the presence or absence of SWS1 and SWS2 did not affect the equiluminant conditions. CONCLUSIONS: RH2 and LWS, but not SWS1 and SWS2, should predominantly contribute to the postreceptoral processes leading to the OMR or, possibly, to luminance detection in general, as the medium-wavelength-sensitive and LWS cones, but not the SWS cones, are responsible for luminance detection in humans.

Changes in a Cone Opsin Repertoire Affect Color-Dependent Social Behavior in Medaka but Not Behavioral Photosensitivity.

Common ancestors of vertebrates had four types of cone opsins: short-wavelength sensitive 1 (SWS1), SWS2, rhodopsin 2 (RH2), and long-wavelength sensitive (LWS) types. Whereas fish and birds retain all the types, mammals have lost two of them (SWS2 and RH2) possibly because of their nocturnal lifestyle during the Mesozoic Era. Considering that the loss of cone opsin types causes so-called color blindness in humans (e.g., protanopia), the ability to discriminate color by trichromatic humans could be lower than that in potentially tetrachromatic birds and fish. Behavioral studies using color-blind (cone opsin-knockout) animals would be helpful to address such questions, but it is only recently that the genome-editing technologies have opened up this pathway. Using medaka as a model, we introduced frameshift mutations in SWS2 (SWS2a and/or SWS2b) after detailed characterization of the loci in silico, which unveiled the existence of a GC-AG intron and non-optic expressed-sequence-tags (ESTs) that include SWS2a in part. Transcripts from the mutated SWS2 loci are commonly reduced, suggesting that the SWS2a/b-double mutants could produce, if any, severely truncated (likely dysfunctional) SWS2s in small amounts. The mutants exhibited weakened body color preferences during mate choice. However, the optomotor response (OMR) test under monochromatic light revealed that the mutants had no defect in spectral sensitivity, even at the absorbance maxima (λmax) of SWS2s. Evolutionary diversification of cone opsins has often been discussed in relation to adaptation to dominating light in habitats (i.e., changes in the repertoire or λmax are for increasing sensitivity to the dominating light). However, the present results seem to provide empirical evidence showing that acquiring or losing a type of cone opsin (or changes in λmax) need not substantially affect photopic or mesopic sensitivity. Other points of view, such as color discrimination of species-specific mates/preys/predators against habitat-specific backgrounds, may be necessary to understand why cone opsin repertories are so various among animals.

Protanopia (red color-blindness) in medaka: a simple system for producing color-blind fish and testing their spectral sensitivity.

BACKGROUND: Color perception is important for fish to survive and reproduce in nature. Visual pigments in the retinal photoreceptor cells are responsible for receiving light stimuli, but the function of the pigments in vivo has not been directly investigated in many animals due to the lack of color-blind lines and appropriate color-perception tests. METHODS: In this study, we established a system for producing color-blind fish and testing their spectral sensitivity. First, we disrupted long-wavelength-sensitive (LWS) opsins of medaka (Oryzias latipes) using the CRISPR/Cas9 system to make red-color-blind lines. Single guide RNAs were designed using the consensus sequences between the paralogous LWSa and LWSb genes to simultaneously introduce double-frameshift mutations. Next, we developed a non-invasive and no-prior-learning test for spectral sensitivity by applying an optomotor response (OMR) test under an Okazaki Large Spectrograph (OLS), termed the O-O test. We constructed an electrical-rotary cylinder with black/white stripes, into which a glass aquarium containing one or more fish was placed under various monochromatic light conditions. The medaka were irradiated by the OLS every 10 nm, from wavelengths of 700 nm to 900 nm, and OMR was evaluated under each condition. RESULTS: We confirmed that the lws - medaka were indeed insensitive to red light (protanopia). While the control fish responded to wavelengths of up to 830 nm (λ = 830 nm), the lws - mutants responded up to λ = 740 nm; however, this difference was not observed after adaptation to dark: both the control and lws - fish could respond up to λ = 820 ~ 830 nm. CONCLUSIONS: These results suggest that the lws - mutants lost photopic red-cone vision, but retained scotopic rod vision. Considering that the peak absorption spectra (λmax) of medaka LWSs are about 560 nm, but the light-adapted control medaka could respond behaviorally to light at λ = 830 nm, red-cone vision could cover an unexpectedly wide range of wavelengths, and behavioral tests could be an effective way to measure spectral sensitivity. Using the CRISPR/Cas9 and O-O systems, the establishment of various other color-blind lines and assessment of their spectra sensitivity could be expected to proceed in the future.