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.

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.

1. A primary objective of marine ecology is to enhance our understanding of the drivers of marine fish population dynamics. These dynamics are readily understood using the framework of metapopulation ecology: for a population to persist, it must be able to self-replicate or be connected to other populations. The drivers of these criteria include rates of reproduction and dispersal, which together determine the rates of recruitment. Recruitment is, however, highly variable, and we do not know widespread patterns and causes of this variation in marine fishes. 2. Reproductive success is notoriously difficult to measure in the wild, and even more so for coral reef fishes that reside underwater. As a proxy, mating success can be predicted from more easily measured determinants, such as variation in individual traits and habitat characteristics. 3. Dispersal has, until recently, been the ‘black box’ of marine ecology, but it can be helpfully described using dispersal kernels. Currently, only five marine fishes have had their dispersal kernel empirically estimated using parentage analyses. From these studies, we see that there are at least four orders of magnitude variation in individual dispersal distances within a species. 4. Many coral reef fishes live in close association with macroinvertebrate hosts, which provide nesting sites and protection from predators. The fish themselves exhibit parental care over clutches of eggs, which hatch into dispersive larvae and recruit back into a suitable host. This life-history suggests habitat quality variation at the scale of the individual host may play a large role in determining relative reproductive and dispersal rates. To explore the role of variation in habitat quality on marine fish reproductive ecology and dispersal ecology, I perform the following three studies: 5. First, I investigate the determinants of variation in mating success in the sponge-dwelling goby, Elacatinus lori. While this fish was the first to have its larval dispersal kernel empirically measured, there were outstanding questions about its reproductive ecology. I describe the characteristics of its breeding habitat, its genetic mating system, and the correlates of male mating success. I show that sponges occupied by breeding males tend to be larger than other sponges, males exhibit a polygynous mating system, and male size, but not sponge size, is positively related to multiple metrics of male mating success. 6. Second, I test for the possibility of plasticity in larval dispersal phenotypes in response to parental habitat quality variation in the orange anemonefish, Amphiprion percula. To pursue this hypothesis, we must first establish that the conditions for parental effects to evolve are met in this system. An initial condition is whether parents can predict the habitat quality that their offspring will encounter. I test for spatial predictability of habitat quality by describing the strength and scale of spatial autocorrelation of three habitat quality indicators: anemone size, female size, and clutch size. I show that all three habitat quality indicators are positively correlated at the scale of an individual patch reef, suggesting that selection might favor parents that increase allocation to offspring that stay within the natal reef if they are in good habitat and increase allocation to offspring that travel farther and leave if they are in poor habitat. Results from this study motivate further investigation of dispersal plasticity in A. percula. 7. Third, I experimentally test the hypothesis that parents can adjust the dispersal-related traits of their offspring in response to changes in habitat quality using a lab population of Amphiprion percula. I describe changes in body size and swimming ability of offspring in response to parental habitat quality by manipulating the parents’ food rations. I find plasticity of larval body size, with parents producing larger offspring when on a low food ration than when on a high food ration. Results from this study motivate future studies to investigate plasticity of larval dispersal distances in the field. 8. In sum, I investigate how variation in habitat quality influences reproductive success, how variation in habitat quality is spatially predictable, and how variation in habitat quality influences dispersal-related traits in two important model systems in marine metapopulation ecology. The work advances our understanding of the role of habitat in determining these vital rates and population dynamics with implications for conservation and fisheries management.

Le potentiel évolutif des populations naturelles à répondre aux changements environnementaux détermine leur capacité à s'adapter et à survivre. Pour achever une évolution adaptative, la fitness doit être héritable, i.e. doit être transmise des parents à leurs descendants par des gènes. Pour pouvoir mesurer le potentiel évolutif d'une population en milieu naturel, il est primordial d'avoir au préalable des informations sur la fitness des individus qui la composent, mais aussi de connaître la fitness de leurs descendants. Les mesures de fitness sont extrêmement rares, en particulier pour les espèces marines, où les relations entre les générations sont rarement connues. Dans cette thèse, je présente le premier pedigree construit pour une population de poissons marins sur la base du suivi génétique mené depuis plus de 10 ans sur les poissons-clowns orange de l'île de Kimbe (Papouasie-Nouvelle Guinée). Le pédigrée comprend 2927 individus et révèle une philopatrie natale sur 5 générations. L'approche en génétique quantitative révèle que la fitness locale a une très faible valeur d'héritabilité (<1%). La variation génétique additive et les effets maternels sont également très faibles (<1%). En revanche, l'habitat est le principal facteur qui explique les différences de fitness locale observées entre les individus (jusqu'à 96,5%). Ensemble, ces résultats suggèrent que, bien que l'environnement impose une forte pression de sélection sur la fitness locale, la population de poissons-clowns orange a un très faible potentiel d'évolution face aux changements environnementaux. / The evolutionary potential of wild populations to respond to environmental change will determine their capacity to adapt and survive. However, in order to achieve evolutionary change, variation in the contribution of an individual to the next generation — its fitness — must have a genetic basis and be heritable. The study of the evolutionary potential of populations requires longitudinal and relatedness data with different environments to partition the contribution of genes, maternal effects and environment on fitness. Estimates of genetic heritability of fitness traits are extremely rare, especially for marine species, where inter-generational relationships are rarely known. Here, we present the first multi-generational pedigree for a marine fish population by repeatedly genotyping all individuals in a population of the orange clownfish (Amphiprion percula) at Kimbe Island (Papua New Guinea) over a 10-year period. Based on 2927 individuals, our pedigree analysis revealed that longitudinal philopatry was recurrent over five generations. We show that local reproductive success has a very low (<1%) but significant heritability. We also show that additive genetic variation and maternal effects on local fitness are both extremely low (<1%). Habitat is the major driver that explain differences in the contribution of individuals to the next generation in the local population (until 96.5%). Together these results suggest that while the environment imposes strong selection pressures on the local fitness, the low heritability indicates the orange clownfish population has little evolutionary potential to adapt to local environmental changes.

The objectives of the study were to determine whether or not behaviour could be used to predict spawning in Amphiprion akallopisos, and to document the behaviour of this species under various environmental conditions in captivity. The spawning behaviour of A. akallopisos was studied and quantified. Three behaviour patterns - belly touching, nest cleaning by the female and mutual nest cleaning (by the male and the female) were identified as predictors for spawning. The reproductive behaviour of A.akallopisos under three photoperiods was investigated. The photoperiods were: 14L:10D, 10hr15minL:13hr45minD and a natural photoperiod cycle condensed into three months. A. akallopisos maintained under 14 L:10D exhibited a significantly higher frequency and duration of chasing, nest cleaning and total interaction compared to fish kept under 10hr15minL:13hr45minD. A photoperiod of 14L:10D was selected for further studies. As manipulation of photoperiod did not induce spawning, GnRHa was administered to the fish in the diet at levels of 10, 20, 40 and 80 μg/kg BW. Control groups were fed untreated food. None of the dosages were successful in inducing spawning, or spawning behaviour in A. akallopisos. It is possible that the method of hormone application was not suitable for this species. It is also possible that behaviour may regulate blood hormone levels as opposed to endocrine status influencing behaviour. In the third trial, A. akallopisos was maintained with, and without sea anemones in order to determine whether or not anemone hosts are necessary for spawning. The presence of anemones did not induce spawning and A. akallopisos kept without anemones exhibited significantly more interactive behaviour than fish kept with hosts. Spawning of A. akallopisos at a later date without anemones suggests that anemones are not necessary for spawning in A. akallopisos. Light intensity was investigated as a cue for spawning. The reproductive behaviour of A. akallopisos was then studied under light intensities of 4.16 x 10¹⁵ quanta.sec⁻¹.cm⁻², 8.85 x 10¹⁵ quanta.sec⁻¹.cm⁻² and this intensity plus natural light. Spawning occurred mainly under 8.85 x 10¹⁵ quanta.sec⁻¹.cm⁻². Fish maintained under this light intensity exhibited significantly more of nest cleaning behaviour in terms of frequency and duration than fish maintained under low light intensity. A minimum light intensity of 8.85 x 10¹⁵ quanta. sec⁻¹.cm⁻² is recommended for conditioning of this species. This study can be of practical relevance to hatchery managers who can use the methods developed and record predictors for spawning in A. akallopisos and other Amphiprion species. In this way imminence of spawning may be estimated. Most importantly, the study also has academic merit as little work has been undertaken in this field. Although the observational method used in this study was adequate for the purposes of the investigation, future work of this nature should incorporate other methods of documenting gonadal development such as gonadal staging and GSI in order to obtain more conclusive results.

The relationship between clownfish and sea anemones is a beloved and iconic example of marine symbiosis. However, ecological information in the eastern Red Sea about the endemic Red Sea clownfish, Amphiprion bicinctus, and its host sea anemones remains relatively incomplete. For example, previous studies report that A. bicinctus forms mutualistic relationships with three to five host anemone species in the Red Sea. But the reported number and combination of host anemone species varies substantially among sources and little is known about host anemone distributions at different scales on the eastern coastline. To better understand the ecology of A. bicinctus and its host sea anemones, we conducted 70 surveys in three regions of Saudi Arabia and one region in Djibouti. We then analyzed distribution patterns for all anemone species observed in these regions, to attain deeper knowledge of anemone habitat usage and relative abundance. We recorded six host sea anemone species associated with A. bicinctus in the Red Sea, one of which represents a new case of symbiosis, and we identified differences in species composition and abundance across different reef scales. We noted patterns of decreasing anemone diversity with increasing latitude, beginning at 20° N, and greater overall anemone abundance in the central Red Sea. We also used field and laboratory observations to examine anemonefish abundance, survival, and fecundity on different species of anemones at different life history stages. In sum, this thesis collectively identifies eastern Red Sea anemone hosts for A. bicinctus, evaluates their regional and fine-scale distributions, and assesses how different anemone species impact their anemonefish occupants on different levels.

Phenotypic plasticity, the capacity of individuals to respond to changing environments by modifying traits, is critically important in allowing biological innovation in the face of environmental change. My dissertation used the clown anemonefish (Amphiprion percula) study system to explore plasticity in parenting strategies in response to variable ecological and social environments. In Part I, I investigated plasticity in response to ecological environment. First, I explored how resource variation influences parenting strategies. I measured parental behaviors in A. percula under two feeding regimes in the laboratory. I demonstrated that clownfish exhibit plasticity in parental care, and that there is significant among individual variation, i.e., personality, in parenting strategies. Second, I tested how plasticity affects life history strategies in the field. I measured habitat, reproductive, and parental traits in a natural population and found positive correlations between resource availability (anemone size) and body size, reproduction, and parental care. I conducted an experimental manipulation of resource availability and found that reproduction and parental care are plastic, providing a causal link between habitat quality variation and reproductive success in natural populations. In Part II, I investigated plasticity in response to social environment. In my third chapter, I explored how parents utilize social information to optimize their parental investment. I developed a game theory model that provides predictions for how power and punishment influence negotiations between parents over offspring care. The model predicts that the threat of punishment by a powerful parent will result in greater partner effort and, as a result, the offspring receive more total care when there is power and punishment in negotiations. Finally, I tested alternative models along with the model I developed, investigating how parents respond to each other to reach a negotiated settlement over offspring care. I experimentally handicapped one pair member and measured the response of the other parent. I found that anemonefish males and females do not respond directly to changes in their partner’s behavior, contrary to predictions of current negotiation models. Together, results from my dissertation extend our understanding of plasticity of parental care, providing a framework for understanding how parents will respond to changing environments.

Videotape and audio recordings of a patch reef in Ant Atoll, Pohnpei, Micronesia were analyzed for the presence of bioacoustic activity produced by species of the teleost family Pomacentridae. The acoustic parameters of fish calls were measured and then identified as belonging to either Amphiprion melanopus or Stegastes nigricans. Visual and statistical methods of analysis were combined to compare the calls of the two species and to test for measurable differences in the acoustic parameters of their calls. Soundscape data showed similar patterns to previously observed tropical reef environments. Fish produced single or multi-pulsed calls in the frequency range of 0-1000 Hz that were often accompanied by observable behaviors such as a chases and displays of dominance. Results indicated that the species on this atoll produce similar calls that cannot be distinguished using acoustic data alone. Visual confirmation was necessary for definitive identification of sound producers and in many cases was not possible beyond the family level. Implications for passive acoustic monitoring are that acoustic data collection alone may be able to capture fish abundance but may not be sufficient to fully capture species richness in regions where populations of closely related fish have similar signals.

Thesis (Ph.D.) - James Cook University, 2004. / Typescript (photocopy) Bibliography: leaves 105-116.

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.