31Vertebrate vision is accomplished through a set of light-sensitive photopigments, which 32 are located in the photoreceptors of the retina and consist of a visual opsin protein bound 33 to a chromophore. In dim-light, vertebrates generally rely upon a single rod opsin (RH1) 34 for obtaining visual information. By inspecting 101 fish genomes, we found that three 35 deep-sea teleost lineages have independently expanded their RH1 gene repertoires. 36 Amongst these, the silver spinyfin (Diretmus argenteus Johnson 1863) stands out as having 37 the highest number of visual opsins known for animals to date (2 cone and 38 rod opsins). 38 Spinyfins simultaneously express up to 14 RH1s encoding for photopigments with 39 different peak spectral sensitivities (λmax=448-513 nm) that cover the range of the residual 40 daylight, as well as the bioluminescence spectrum present in the deep-sea. Our findings 41 present novel molecular and functional evidence for the recurrent evolution of multiple 42 rod opsin-based vision in vertebrates. 43 44 SHORT ABSTRACT: Contrary to the single rod opsin used by most vertebrates, some fishes 45 use multiple rod opsins for vision in the dimly lit deep-sea. 46 Animals use vision for a variety of fundamental tasks including navigation, food acquisition, 47
Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of eggs at the one-cell stage was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 50% of injected eggs. Further analysis of the subcloned mutant gene sequences revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in G0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful resource for future experimental studies that aim to elucidate gene function in anemonefishes and reef fishes in general.
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