Seeing Nemo: molecular evolution of ultraviolet visual opsins and spectral tuning of photoreceptors in anemonefishes (Amphiprioninae)

Mitchell, Laurie J.; Cheney, Karen L.; Chung, Wen-Sung; Marshall, N. Justin; Michie, Kyle; Cortesi, Fabio

doi:10.1101/2020.06.09.139766

Cited by 5 publications

(9 citation statements)

References 104 publications

(118 reference statements)

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“…Anemonefish have interesting features, such as plasticity in sex differentiation (Deangelis & Rhodes, 2016), symbiotic relationships with sea anemones (Nedosyko et al., 2014; Titus et al., 2019), and parental care of their embryos. Furthermore, their color variation and pigment pattern formation are interesting topics for researchers (Bell et al., 1982; Maytin et al., 2018; Mitchell, Cheney, et al., 2020; Moyer, 1976; Salis et al., 2018, 2019). Among anemonefish, A. ocellaris has been well known as a popular aquarium fish that can be reared in small‐scale facilities.…”

Section: Introductionmentioning

confidence: 99%

Establishment of culture and microinjection methods for false clownfish embryos without parental care

et al. 2021

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Anemonefish, including the false clownfish Amphiprion ocellaris, are attractive model organisms because of their unique features, such as sex change and brilliant color patterns in mutants. However, anemonefish are not widely used to study gene function using reverse genetic approaches owing to microinjection difficulties and subsequent rearing and hatching of embryos without parental care. A. ocellaris embryos are spawned on a hard substrate and cared for by their parents until hatching. However, the eggs need to be detached from the substrate and raised without their parents to perform successful microinjection. We established a method to culture and hatch A. ocellaris embryos without spawning substrates or parental care. We found that changing water and generating water flow are critical for culturing the embryos, and that water flow (as physical stimulation) and complete darkness in the dark period are necessary for successful hatching. We further investigated the effectiveness of microinjection into the yolk sac of fertilized eggs rather than into the cytoplasm, which makes microinjection easier. A reporter RNA injected into the yolk sac was transferred to the cytoplasm and translated, indicating that yolk sac microinjection is an efficient alternative as has been used for zebrafish. These findings highlight the potential of A. ocellaris as an experimental model organism for reverse genetics, and our methods could be applied to other anemonefish species.

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Section: Introductionmentioning

confidence: 99%

Establishment of culture and microinjection methods for false clownfish embryos without parental care

et al. 2021

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“…2A, B). The gene sequence for A. ocellaris RH2B was obtained from a previous study (Mitchell et al 2020), and the same approach described by Mitchell et al 2020 was used to identify the tyr gene sequence in the A. ocellaris genome (Tan et al 2018). All gene sequences were viewed in Geneious (v.2019.2.3), and the “Find CRISPR Sites…” function was used to screen suitable sgRNA sequences with search parameters that included a target sequence length of 19-bp or 20-bp, an ‘NGG’ protospacer-adjacent-motif (PAM) site located on the 3’ end of the target sequence, and off-target scoring against the A. ocellaris genome (see supplementary material for a list of sgRNA sequences).…”

Section: Methodsmentioning

confidence: 99%

“…The fascinating aspects of anemonefish biology has led to their use in multiple areas of research including for studying the physiological responses of reef fishes to the effects of climate change (Scott & Dixson, 2016; Beldade et al 2017; Norin et al 2018), the hormonal pathways that regulate sex change (Casas et al 2016; Dodd et al 2019) and parental behaviour (DeAngelis et al 2017, 2018; Iwata & Suzuki, 2020), and the physiological adaptations for group-living (Buston, 2003; Buston & Cant, 2006). Moreover, anemonefishes are being used to understand the visual capabilities of fish (Stieb et al 2019; Mitchell et al 2020) and evolution of skin colour diversity (Maytin et al 2018; Salis et al 2018; Salis et al 2019) in reef fishes. However, despite the wide-reaching applications of anemonefish research, the genetic basis for many of their traits remain to be empirically investigated.…”

Section: Introductionmentioning

confidence: 99%

“…Inherent challenges specific to gene-editing anemonefishes and many other demersal spawning reef fishes include the injection and/or care of their substrate-attaching eggs (Roux et al 2019) and pelagic larval stage (Leis & McCormick, 2002). Therefore, a protocol for performing CRISPR/Cas9-mediated genome editing in anemonefishes would be highly beneficial for diverse areas of research to directly test candidate genes facilitating sex change (Dodd et al 2019), colour vision (Mitchell et al 2020) and skin pattern development (Salis et al 2019).…”

Section: Introductionmentioning

confidence: 99%

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CRISPR/Cas9-mediated generation of biallelic G0 anemonefish (Amphiprion ocellaris) mutants

Mitchell

Tettamanti

Marshall

et al. 2020

Preprint

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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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“…To demonstrate the display's use, we conducted colour detection tests with the anemonefish Amphiprion ocellaris , a species with known UV‐sensitive cones ( λ max = ~386 nm) based on molecular and anatomical analyses (Figure ; Mitchell et al., 2020) and spatial acuity <5.80 cycles/degree (Stieb et al., 2019). The behavioural experiment was conducted between November 2019 and February 2020 under the approval from The University of Queensland's Animal Ethics Committee (SBS/077/17).…”

Section: Behavioural Experimentsmentioning

confidence: 99%

A five‐channel LED display to investigate UV perception

et al. 2021

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1. The ability to see ultraviolet (UV) light (<400 nm) may have importance for foraging, communication or navigation in many taxa including insects, crustaceans, fishes, amphibians, reptiles, birds and mammals. Behavioural experiments reveal how vision mediates such behaviour; however, our knowledge of UV perception is constrained by the challenge of creating and calibrating stimuli that reflect or emit UV. Commonly used technologies for displaying visual stimuli-such as computer screens and printers-are designed for human vision and thus are unsuitable for testing UV perception. 2. To overcome this limitation, we designed and constructed a display with five spectral channels with peak wavelengths as follows: red at 629 nm, green at 526 nm, blue at 466 nm, violet at 395 nm and UV at 367 nm. Each pixel of the display consists of five LEDs with a baffle to prevent crosstalk from adjacent pixels and a diffuser to promote uniform colour mixing. The LEDs are driven by high-performance pulse-width-modulated constant-current drivers with a maximum flicker rate of 64 kHz and a maximum frame rate of 6.5 kHz. This method allows colour mixing with wavelengths as low as 350 nm to be calibrated and tested rapidly and concurrently. 3. To demonstrate the utility of this display, we conducted colour detection tests using the anemonefish, Amphiprion ocellaris, a species known to have UV-sensitive cones. Fish were able to associate pecking all target colours ('Blue', 'UV-grey' and 'UV') with a food reward, demonstrating for the first time, UV perception in A. ocellaris. 4. The RGB-V-UV LED display is a useful device for behavioural tests of colour vision across a broad spectrum (350-650 nm) visible to many animals and can be used to investigate various questions concerning animal perception, including colour discrimination and categorisation. We include design documents and source code so this system can be further developed and modified to investigate other visual behaviours in a variety of taxa. K E Y W O R D S animal behaviour, colour vision, coral reefs, ultraviolet vision, visual ecology This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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Seeing Nemo: molecular evolution of ultraviolet visual opsins and spectral tuning of photoreceptors in anemonefishes (Amphiprioninae)

Cited by 5 publications

References 104 publications

Establishment of culture and microinjection methods for false clownfish embryos without parental care

Establishment of culture and microinjection methods for false clownfish embryos without parental care

CRISPR/Cas9-mediated generation of biallelic G0 anemonefish (Amphiprion ocellaris) mutants

A five‐channel LED display to investigate UV perception

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