As anemonefishes of the genus Amphiprion are emerging as a model organism for marine science, it is critical to have standardization in housing and husbandry protocols for research laboratories. Here, we conducted a literature review, a questionnaire survey, and two experiments regarding the housing and husbandry of anemonefishes for use in research. The literature review identified the need for standardization and optimization of multiple variables including tank volume, diet composition, and lighting type for the housing of broodstock pairs. The questionnaire survey was completed by 27 laboratories, with a 45% response rate, across 11 different countries in Europe, North America, Asia, and Australia. Results from the survey confirmed the need for standardization or optimization of laboratory protocols. The experimental manipulation of housing of Amphiprion percula was conducted over a 3-month period, where reproduction was monitored for 8 breeding pairs housed in large, 120-liter (30-gallon) tanks versus 7 breeding pairs housed in small, 9-liter (2-gallon) tanks. Results suggest that reproductive output was significantly reduced among breeding pairs in experimental 9-liter tanks when compared to those housed in standard 120-liter tanks. The experimental manipulation of husbandry of A. percula was conducted over a 3-month period, where reproduction was monitored across 19 breeding pairs on a high ration diet (1 mL scoop of pellets) versus 15 breeding pairs on a low ration diet (0.5 mL scoop of pellets). Results suggest that reproductive output was significantly reduced among breeding pairs on a low ration diet when compared to those on a high ration diet. Although the experimental 9-liter tanks were likely below the optimum for reproductive output, the fact that reproduction still occurred suggests that the fish will reproduce in tanks smaller than 120 liters and that 20-75 liter tanks may be the best option for housing large numbers of A. percula and similar species. The reproductive output of broodstock pairs fed a high ration diet is similar to that seen in the wild, and provision of higher quality foods (e.g., cuttlefish, shrimp, fish eggs, etc.) beyond commercial pellets may enhance survival of larvae. In conclusion, our research suggests that the anemonefish research community should be discussing the optimization and standardization of housing and husbandry or, minimally, recognize that housing and husbandry influence a wide range of traits and will influence the results and conclusions drawn from experiments.

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Larval dispersal is one of the most complex phenomena that connects populations when larvae migrate over long distances from their native reefs to a new one. Dispersal outcomes are largely variable among individuals, even siblings, and the factors that drive this variability are not understood, making it challenging to create protection plans for marine species. Clownfish, Amphiprioninae, are among the most important coral reef species economically. Thus, creating protection plans for clownfish is necessary, especially as they are under habitat loss threat. Clownfish are considered an optimum model of organisms for studying larval dispersal and population connectivity mainly due to their relatively short larval dispersive phase and ease of breeding them in captivity. Since dispersal traits are influenced by genes, transcriptomic studies can reveal insights into larval dispersal. Thus, the variation in dispersal outcomes could be better understood if the differential expression of genes related to dispersal, such as swimming abilities, are considered. However, there is a lack of knowledge in the basics of gene expression in clownfish larvae. The aim in this thesis is to explore the ontogenic variability in RNA quantities across development in clownfish larvae as a first step of understanding the nature of their gene expression and optimizing RNA extractions and quantifications methods using clownfish as a model organism. We performed an RNA quantification study using larvae of Amphiprion bicinctus clownfish reared at KAUST SeaLabs. This thesis provides some insights regarding the pooled and individual quantities of RNA across time. The optimal pool size and age for various transcriptomic studies was determined. It was concluded that extracting RNA from young larvae is difficult. Depending on the research, it might be necessary to use older larvae or other techniques. The results from this thesis will build the foundation needed as a first step to perform differential gene expression studying the causes of variability in dispersal traits in A. bicinctus and the link between these traits and transcriptomics.

Long-term studies are important for understanding the intricacies of population dynamics over time. Self-recruitment and social hierarchy are valuable tools to quantify the rates at which populations change. In mutualistic symbiosis, where two species benefit from the relationship, different selective pressures and life histories can have unintended consequences on the population dynamics of both species. Anemonefish live in a sized-based hierarchy where individuals queue to be part of the breeding pair (ranks 1 and 2). They have a mutualistic association with their host anemone; the identity of the anemone can impact their growth and fecundity. However, there is limited knowledge on the anemone lifespan and its site persistence over time. Here, we investigate rank changes and self-recruitment in Amphiprion percula and persistence in a common host anemone, Stichodactyla gigantea, on the remote island of Kimbe Island in Papua New Guinea. The populations of A. percula (n = 1,530) and their local host anemones, S. gigantea (n = 290) and Heteractis magnifica (n = 174), were sampled exhaustively in 2011 and 2019. Using DNA profiling, I determined the fate of individuals between years. We found that 21% of the A. percula population survived over the eight-year time gap compared to the 69% survival of the associated S. gigantea population in a six-year time gap. Half of the surviving A. percula individuals increased in rank and exhibited faster growth rates living on S. gigantea compared to H. magnifica. Self-recruitment was high in both years, 47% in 2011 and 39% in 2019, with one individual returning to its natal anemone. Our findings provide rare insights into one of the most charismatic symbiotic relationships in the marine environment such as the first documentation of longevity in a host anemone.

Lunar cycles of reproduction are a widespread phenomenon in marine invertebrates and vertebrates. It is common practice to infer the adaptive value of this behavior based on the population level pattern. This practice may be flawed if individuals within the population are employing different reproductive strategies. Here, we capitalize on a long-term field study and a carefully controlled laboratory experiment of individually identifiable clown anemonefish, Amphiprion percula, to investigate the individual reproductive strategies underlying population-level patterns of reproduction. The field data reveal that A. percula exhibit a lunar cycle of reproduction at the population level. Further, the field data reveal that there is naturally occurring variation among individuals and within individuals in the number of times they reproduce per month. The laboratory experiment reveals that the number of times individuals reproduce per month is dependent on their food availability. Individuals are employing a conditional strategy, breeding once, twice or thrice per month, depending on resource availability. Breaking down the population level pattern by reproductive tactic, we show that each reproductive tactic has its own non-random lunar cycle of reproduction. Considering the adaptive value of these cycles, we suggest that all individuals, regardless of tactic, may avoid reproducing around the new moon. Further, individuals may avoid breeding in synchrony with each other, because of negative frequency dependent selection at the time of settlement. Most importantly, we conclude that determining what individuals are doing is a critical step toward understanding the adaptive value of lunar cycles of reproduction.

Parental care is a critical component of many breeding systems that enhances both parent and offspring fitness. It is widely recognized that parental care varies in quality and quantity in response to variation in offspring need, parental condition, and ecological context. One context that has been underexplored, however, is the difference in parental care between day and night, with many parental care studies focused on diurnal care. To fully understand the evolution of parental care, the costs and benefits and the negotiations between parents, both diurnal and nocturnal care must be accounted for. Here, we show that nocturnal parental care exists in the clownfish Amphiprion percula, with males and females actively caring for offspring at night throughout embryo development and during hatching in a laboratory setting. At night, as during the day, males are the dominant caregivers and both parents increase care as embryos mature. At night, in contrast to the day, females provide substantially more care. This increase in energy expended by parents on care may be a mechanism to increase dissolved oxygen in the water at night, improving embryo survival. We also document the existence of three new nocturnal parental care behaviors, not previously seen during the day: vigorous fanning, vigorous mouthing, and anemone biting. These behaviors are particularly frequent during hatching, and although they are not necessary for hatching to occur, they may serve to enhance the embryos’ successful transition from the benthos to the water column. This study contributes to our understanding of parental care, demonstrating the importance of considering nocturnal care if we are to gain a complete understanding of the evolution of parental care. / 2023-03-16T00:00:00Z

Coral reef fish are known to have a bipartite life cycle: a dispersive pelagic larval phase (DPL), where larvae ‘travel’ away from their parents to occupy new habitats, and a sedentary reef phase characterized by juveniles and adults. Study of the DPL is an essential prerequisite to understand how persistence, connectivity, and gene flow operate between populations. Clownfish have been the subject of considerably varied fields of research in recent decades, not only due to their economic value, but also because of their ease of maintenance in laboratory settings, regular spawning, and short DPL. Their life cycle is also defined as bipartite, with a DPL lasting around 15 days until settlement, and the fish exhibit a symbiotic relationship with sea anemones. The transitions between phases are associated with a series of morphological, behavioural, genetic, cellular and molecular changes. However, these changes are poorly characterized in some species of clownfish. Therefore, the Red Sea clownfish, Amphiprion bicinctus, was reared to fill the gap in the literature by investigating their i) morphology, ii) swimming abilities, and iii) differential gene expression (DGE) throughout ontogeny. The DPL was characterized in the preflexion, flexion and postflexion stages, according to flexion of the notochord, on 0, 2 and 10 days post hatch (dph), respectively. Metamorphosis was defined by a morphological transition between the DPL and juvenile phases, associated with the appearance of white stripes, on day 10 and until the end of the experiment, on day 14. Additionally, metamorphosis is followed by a behavioural change at 14 dph, called settlement, where larvae transition from free swimming in the water column to associating with the substrate. We also demonstrate that the larvae present active swimming from the moment they hatch, and the swimming speed increases throughout DPL. Overall, the development of A. bicinctus is similar to other clownfish species. This work provides a strong baseline on the development of A. bicinctus, which may open the door to a deeper understanding of the role of dispersal behaviours and enhance our ability to design effective marine reserve networks in the Red Sea.