The false clown anemonefish Amphiprion ocellaris is a prominent icon of the coral reef. Anemonefish are famous for their mutualistic symbiosis with sea anemones, popularity in the marine aquarium hobby, and presence in popular culture. Amphiprion species also serve as important model organisms in the life sciences for studying a variety of biological processes such as larval recruitment, ultraviolet vision, monogamy, female behavioral dominance, protandrous sex change, paternal care, and mutualistic symbiosis with anemones (Beldade et al., 2016, Fricke and Fricke, 1977, Laudet and Ravasi, 2022, Mitchell et al., 2021, Buston and García, 2007, Mitchell et al., 2024). A. ocellaris live in small groups with a small home range immediately surrounding their host sea anemone. This ecological feature provides a number of logistical advantages for using anemonefish in laboratory and field research. In the laboratory, anemonefish are comfortably housed in modestly sized aquaria, and readily display natural physiology and behavior including protandrous sex change (Dodd et al., 2019, Parker et al., 2023, Parker et al., 2022, Madhu et al., 2010) and parental care (DeAngelis and Rhodes, 2016, Phillips et al., 2020, DeAngelis et al., 2017, DeAngelis et al., 2020, Ghosh et al., 2012, Barbasch et al., 2022). Additionally, larvae are easily reared in aquaria, meaning their entire life cycle can be studied under experimentally controlled conditions in the lab (Mitchell et al., 2021, Madhu et al., 2006, Roux et al., 2021, Laudet and Ravasi, 2022).
Recently, we developed methods for gene knockout in A. ocellaris using CRISPR/Cas9 technology (Mitchell et al., 2021). Briefly, we injected CRISPR/Cas9 reagents into A. ocellaris eggs, reared larvae bearing the null allele to adulthood, and genotyped them to confirm the gene had been knocked out. While these methods opened the door for genetic manipulation in a single anemonefish, we did not confirm that the mutation was present in the germ cells, and did not attempt to found mutant lines for longer-term studies.
Continued use of the anemonefish as a model organism will require methods for stable germ line transmission and methods for inserting genes into the genome. This includes inserting genes from other species or modified genes, also referred to as transgenesis. Transgenes can be selectively expressed in certain cells by using the promoter of a gene of interest to drive their expression. This approach is often used to visualize cell populations that express a gene by using the promoter of that gene to control the expression of a reporter such as green fluorescent protein (GFP) or lacZ (Ma et al., 2015). Other purposes include inserting genetic constructs that allow the conditional knockout or spatiotemporal control over expression of specific genes (e.g., cre-lox, doxycycline) (Campbell et al., 2012, Felker and Mosimann, 2016). Transgenes expressing channel rhodopsin (Seki et al., 2023), or designer chemoreceptors can be used to manipulate the activation of specific cell types (Silic and Zhang, 2021), and transgenes encoding designer calcium indicators (e.g., GCaMP) may be used to measure cellular activity (Muto et al., 2011). The diversity and capabilities of genetic tools available are expanding rapidly, thus it is important and timely to develop methods in anemonefish for transgene insertion.
While CRISPR/Cas9 technology has been used to insert genes by cut and replace, its reliability and efficiency can be low in certain cell types and organisms, making it challenging to achieve desired gene insertion without extensive screening and selection processes (Rozov et al., 2019). In contrast, the medaka (Oryzias latipes) derived transposon Tol2 provides one of the most efficient methods to date for transgene insertion and is routinely used in zebrafish (Danio rerio) resulting in up to 50% of injected embryos containing the transgene in the germline (Kawakami, 2007). In addition, Tol2 has been successfully adapted for use in several non-model organisms making it an ideal candidate for use in A. ocellaris (Fujimura and Kocher, 2011, Juntti et al., 2013, Stahl et al., 2019).
While methods for Tol2-mediated transgenesis have been established in other non-model organisms, attempting transgenic engineering in A. ocellaris brings new challenges, specifically regarding the microinjection of eggs laid on a solid substrate and high embryonic to larval mortality not found in other species (Yamanaka et al., 2021). Moreover, establishing stable transgenic lines that can be used for longer-term studies has never been accomplished in A. ocellaris or any coral reef fish to our knowledge. Here, we describe methods for the generation of the first stable transgenic line of anemonefish. As proof of principle, we used the Tol2 transposon system to create several lines of A. ocellaris that express green fluorescent protein (GFP) under the control of the ubiquitous promoter for elongation factor-1 α (Ef1α). We describe methods for microinjection, artificial incubation, larval rearing, and transgene detection for future use in transgenic line development in A. ocellaris.