Molecular Evolution of Ultraviolet Visual Opsins and Spectral Tuning of Photoreceptors in Anemonefishes (Amphiprioninae)

Abstract

Many animals including birds, reptiles, insects, and teleost fishes can see ultraviolet (UV) light (shorter than 400 nm), which has functional importance for foraging and communication. For coral reef fishes, shallow reef environments transmit a broad spectrum of light, rich in UV, driving the evolution of diverse spectral sensitivities. However, the identities and sites of the specific visual genes that underly vision in reef fishes remain elusive and are useful in determining how evolution has tuned vision to suit life on the reef. We investigated the visual systems of 11 anemonefish (Amphiprioninae) species, specifically probing for the molecular pathways that facilitate UV-sensitivity. Searching the genomes of anemonefishes, we identified a total of eight functional opsin genes from all five vertebrate visual opsin subfamilies. We found rare instances of teleost UV-sensitive SWS1 opsin gene duplications that produced two functionally coding paralogs (SWS1α and SWS1β) and a pseudogene. We also found separate green sensitive RH2A opsin gene duplicates not yet reported in the family Pomacentridae. Transcriptome analysis revealed false clown anemonefish (Amphiprion ocellaris) expressed one rod opsin (RH1) and six cone opsins (SWS1β, SWS2B, RH2B, RH2A-1, RH2A-2, LWS) in the retina. Fluorescent in situ hybridization highlighted the (co-)expression of SWS1β with SWS2B in single cones, and either RH2B, RH2A, or RH2A together with LWS in different members of double cone photoreceptors (two single cones fused together). Our study provides the first in-depth characterization of visual opsin genes found in anemonefishes and provides a useful basis for the further study of UV-vision in reef fishes.

Keywords: anemonefishes; gene duplication; reef fish vision; spectral tuning; ultraviolet; visual opsins.

Fig. 1.

Summary of anemonefish visual opsin genes and their synteny. (A) Visual opsin genes mapped on the species tree (modified from Tang et al. [2021]) along with their range of estimated peak spectral absorbance (λ_max) values. A detailed opsin gene phylogeny is presented in supplementary figure 2, Supplementary Material online. (B) Synteny of anemonefish visual genes according to the chromosomal arrangement in Amphiprion percula, including opsin gene coding regions (boxes depict single exons) and their flanking genes (black). Areas highlighted in yellow indicate regions where recombination occurred between opsin gene paralogs. Opsin gene acronyms stand for: RH1, rhodopsin 1 (rod opsin); RH2, rhodopsin-like 2; SWS2, short-wavelength-sensitive 2; LWS, long-wavelength-sensitive; SWS1^†, short-wavelength-sensitive 1/short-wavelength-sensitive pseudogene. *Opsin genes mapped from raw transcriptome reads of the A. akindynos retina (from Stieb et al. [2019]). Image credit: A. nigripes, Ewa Barska via Wikimedia Commons; A. polymnus, Jens Petersen via Wikimedia Commons; A. bicinctus, Patryk Krzyzak via Wikimedia Commons; A. frenatus, Vincent Chen via Wikimedia Commons; A. ocellaris and A. akindynos, Valerio Tettamanti via direct permission.

Fig. 2.

Relative cone opsin expression levels in the retina of captive Amphiprion ocellaris (N = 4; two females and two males) kept under aquarium lighting (see supplementary fig. 1, Supplementary Material online, for illumination spectra). Lines represent the mean proportion of opsin expression relative to the total opsin expression levels of single cones opsins (SWSs) and double cone opsins (RH2s, LWS), respectively. SWS1, short-wavelength-sensitive 1; SWS2, short-wavelength-sensitive 2; RH2, rhodopsin-like 2; LWS, long-wavelength-sensitive. Image credit: Amphiprion ocellaris via direct permission from Valerio Tettamanti.

Fig. 3.

Double-labeling in situ hybridization of expressed opsin mRNAs in retinal single cone (A–D) and double cone (E–L) photoreceptors in adult Amphiprion ocellaris. (A–D) SWS1β (magenta) and SWS2B (yellow) mRNA were often coexpressed in the same single cones. (E–H) RH2A (yellow) and RH2B (magenta) mRNA were expressed in opposite members of double cones across the retina. (I–L) RH2A (yellow) and LWS (magenta) mRNA were expressed in the same double cone members. Representative single and double cones are outlines with a white circle and white ovals, respectively. BF, bright field. Scale bars = 10 µm.