Labrus bergylta (ballan wrasse) have recently emerged as a key resource to aquaculture through proven efficacy in controlling infestations of sea lice (Leclercq et al., 2014a). However, due to complex ecology, and a complete lack of sexual dimorphism gender identification endures as a key restriction to optimising broodstock management therefore male selection and establishing optimal sex ratios is difficult (Talbot et al., 2012). L. bergylta, are noted to demonstrate unusually coloured plasma ranging in hue from green to blue with the haem catabolite biliverdin established as the causal pigment in the majority of cases (Abolins, 1961). As most vertebrates excrete biliverdin, or rapidly metabolise it to prevent toxicity, accumulation to such excess is a phenomenon which merits attention. Notably, correlation between plasma biliverdin and gender has been reported in some Labridae. Although patterns vary between species, the abundance or characteristics were such that sexual identity could be established (Gagnon, 2006). Pigment analysis was therefore proposed as a potential sex-marker in L. bergylta. In the initial experimental phase (Chapter 3), the ultimate aim was to isolate and identify the blue pigment from L. bergylta plasma, and to develop a method of quantification. The initial phase confirmed the target pigment was biliverdin IXα by visible spectroscopy, TLC, HPLC, MSMS, and a series of reactions. Following this, a protocol was developed (Chapter 2) to quantify the pigment. This method was applied accross plasma sampled from four geographically distinct wild populations with established biometrics including age, mass, length, gender and external phenotype. Subsequent analysis revealed that although pigment abundance did not vary relative to ontogeny, and there was no difference in concentration between the binary genders, plasma biliverdin was depleted in individuals undergoing sex change. Although this conclusion was complicated by significant biliverdin variation relative to origin and phenotype, which were interrelated based on relative distributions across populations, further analysis of plasma pigment in related species identified that biliverdin accumulation was associated with protogynous species. Considering the anti-oxidant capacity of biliverdin and other potentially relevant functions, this was indicative of association with the tissue remodelling processes which accompany inversion. During Chapter 3 it was noted that the biliverdin appeared tightly bound to a protein moiety. Based on the hypothesis that the pigment was actively managed and accumulated in L. bergylta plasma by this association, the next phase of experiments (Chapter 4) was an exploration of biliverdin and its binding protein in L. bergylta. The experiments revealed plasma biliverdin comigrated with the protein such that it was depleted from solution at the same rate indicating that all of the pigment was associated. Subsequent electrophoretic experiments using the fractionation products supported this, and UV fluourescence identified fragments of interest in the 25-28 kDa region. To confirm observations from the previous cross species comparison, the study was similarly expanded to include other Labrini. This revealed that although the 25 kDa band was common to all species, and genders, the 28 kDa band was collocated with the protogynous, and as such hyperbiliverdinaemic species. The 28 kDa band was sequenced using MSMS, and was identified as similar to the lipocalin Apolipoprotein A1. In combination with the properties of biliverdin, and considering that ApoA1 is analogous to serum albumin in many telesots, this supported the chromoprotein association as the main mechanism of biliverdin accumulation in such species. Further to the proposed function of biliverdin with inversion processes, and considering relevant literature, the active properties of ApoA1 suggested additional associations with prolonged altered states of metabolism which considering the ecology of L. bergylta would include gender transition, overwintering torpor and prolonged micronutrient limitation, all of which occur simultaneously. Other potential roles include modulating inflammatory responses, inhibiting pathogenic incursions and acting as an external point of contact innate immune response. From this, it was concluded that the data fully supported the previous assertions of biliverdins relevance in protogynous species, and identified a number of properties which could be of great interest to the industry in terms of welfare. The final experimental phase (Chapter 5) had two main aims. The first was to establish whether protogynous inversion could be artificially induced in L. bergylta as a means of generating male fish, and whether size had any effect on the process. The second was then to utilise controlled induction for tracking biliverdin mobilisation across the process to test the previous hypothesis. The preliminary trial demonstrated that both androgen inhibition and non-aromatisable testosterone could stimulate inversion in female L. bergylta. From this, the second trial then determined that although there was a dose dependant effect in that high androgen dosages appeared to compress the inversion process, relative size was not a factor. Gonad histology was used to create a unified scale of protogynous transition which could be expressed as a gradient to structure the biliverdin analysis. Although the biliverdin data demonstrated cryptic trends at the higher resolution gender scales, when the endpoint was condensed back to the binary gender scale employed previously (Chapter 3), the prior assertion of depletion during transition, and therefore the association with sex change associated tissue remodelling was supported. Ultimately this thesis revealed links between the biliverdin macromolecule and the highly unusual metabolic and physiological demands of gender transition in sequentially protogynous hermaphroditic temperate wrasse species.

Cleaning symbioses are a well-studied mutualism among marine species. However, the interactions occurring between cleaner fish and sharks are lacking in research, which makes it a target for further investigation. With this study, intentions were to analyse the behaviour of two kinds of cleaner species, bluestreak cleaner wrasse (Labroides dimidiatus) and moon wrasse (Thalassoma lunare), to be able to distinguish differences in cleaning behaviour on pelagic thresher sharks (Alopias pelagicus). A total of 68,4 hours of video was recorded on the edge of a seamount outside of Malapascua, called Monad Shoal, during 18 days in January 2018. The number of interactions were divided into two categories, where the behaviour was classified as an inspection or a bite and could occur on different patches of the sharks’ body (head, gills, body, dorsal, pectoral, pelvic or caudal fin). In total 118 events occurred which comprised in total 4079 interactions from the two cleaner species. Of these interactions 3626 were considered inspections and 453 were bites. Bluestreak cleaner wrasse conducted 3598 of the inspections and 28 of the inspections were conducted by the moon wrasse. All bites were conducted by bluestreak cleaner wrasses. The results indicated a preference in patches of the body to inspect, where the pelvis got the most inspections on 34,1 %, followed by the pectoral fins on 22,8 %. The dorsal fin and the gills accounted for the least number of bites, with 1,3 % on the dorsal and 1,4 % on the gills. Furthermore, a difference in inspected patches between males and females were discovered, where females got significantly more inspections on their head, gills, body, dorsal and pectoral fin. The pelvis and caudal fin did not show any significant differences.

Hogfish (Lachnolaimus maximus; Walbaum 1792) from Southeast Florida were aged using sectioned otoliths and growth rates were calculated using the von Bertalanffy growth equation. The samples were collected from Broward County (n=209); other regions of Southeast Florida (n=18), the Florida Keys (n=35) and Bahamas (n=43). Growth rates were determined for each of these areas and were then compared to previously reported growth rates from other regions including the eastern Gulf of Mexico and Florida Keys. There was significant separation at the 95% confidence level between growth rates from each reagion. The average maximum fork length increased, from the Florida Keys (336mm) to Southeast Florida (414-mm) by 78-mm. However, the annual survival rate was the same (S=61%) between these two regions and the maximum age of Southeast Florida (age 12) was still half that of the previously reported eastern Gulf (age 23). Broward County was divided into three reef zones each at different depths (5-m, 10-m, and 20-m) and growth rate and survival rate were compared between zones. Results showed a decrease in maximum fork length with reef depth (857-mm, 420-mm, 352-mm), as well as an increase in mean age (age 3, 4, 5), maximum age (9, 10, 12), and survival (42%, 65%, 73%), respectively. The decrease in observed growth rate of an area as a whole (e.g. Florida Keys) may represent an example of Lee’s phenomena caused by increased top-down selective fishing pressure. However, the growth rates of individual hogfish are most likely a result of differences in habitat and food resource availability. This study provides baseline age and growth information for hogfish in Southeast Florida prior to the recent changes to the fishery regulations, which will help fisheries management better understand the effects of alternative management strategies.

Genetic variation was examined in bluehead wrasse, Thalassoma bifasciatum, populations along the Florida Keys. Interpretion of mean heterozygosity (H), percent polymorphic loci per population (P), genetic similarity, and F and G statistics demonstrated a clear division of wrasse populations into "northern" and "southern" groups. Correlation and cluster analyses indicated the six reefs can be grouped in a similar fashion based on genetic and environmental data. The conclusion from this analysis of wrasse populations in the Florida Keys is that substantial population subdivision occurs in response to differing selective pressures created by heterogeneous environments.

Cleaner fish, including ballan wrasse (Labrus bergylta) have been proposed as a sustainable solution to sea lice infestations affecting farmed Atlantic salmon (Salmo salar) globally. However, in order to become sustainable, ballan wrasse need to be farmed. This thesis investigated the establishment of captive broodstock and protocols to optimise hatchery performance and productivity of ballan wrasse. High throughput sequencing was used to develop a panel of novel single nucleotide polymorphic markers (SNPs). These SNPs were used to investigate the phylogeographic structuring of ballan wrasse populations within northern geographic ranges including the UK and Norway. Results indicated fine scale population structuring within the UK suggesting that founder broodstock should be sourced locally to minimise the risk of genetic introgression with wild ballan wrasse. Secondly, captive breeding was benchmarked from harems to determine total egg production over the spawning season. Data quantified the spawning periodicity and seasonal changes in egg quality parameters. In addition, microsatellite markers identified the parental contribution to each spawning event of captive broodstock. Results confirmed, for the first time, the repeat-batch spawning behaviour and suggested that spawning events were single-paired matings. Furthermore, bottlenecks in commercial production were investigated including the benthic adhesive eggs and complex spawning behaviours of ballan wrasse within broodstock tanks. Experiments were conducted to optimise the spawning dynamics and egg productivity using fragmented spawning zones and coloured substrates. Finally, an effective bath treatment for removal of the adhesive gum layer of eggs using the proteolytic enzyme alcalase® was found to assist in egg disinfection and incubation. Overall, this research provides important baseline data on the management of broodstock and the optimisation of hatchery protocols to improve the commercial productivity and performance of ballan wrasse for use as a biological control of sea lice of farmed Atlantic salmon.

Elevated levels of trace elements in the environment are of great concern because of their persistence, and their high potential to harm living organisms. The exposure of aquatic biota to trace elements can lead to bioaccumulation, and toxicity can result. Furthermore, the transfer of these elements through food chains can result in exposure to human consumers. Sea-fill or coastal fill sites are among the major anthropogenic sources of trace elements to the surrounding marine environment. For example, in the Maldives, Thilafushi Island is a sea-fill site consisting of assorted municipal solid waste, with multiple potential sources of trace elements. However, there is limited data on environmental trace element levels in the Maldives, and although seafood is harvested from close to this site, there is no existing data regarding trace element levels in Maldivian diets. Following the Christchurch earthquakes of 2011,

Ultraviolet light is damaging but fish have evolved protective mechanisms, which allows them to live in shallow water reefs, high in UV radiation. This thesis details my investigation into the physiological ecology of solar ultraviolet (UV) absorbing compounds, known as mycosporine-like amino acids found in the external epithelial mucus, and examines the supporting role potentially played by a UV-induced DNA repair mechanism in coral reef fish of the Indo-Pacific. Using reverse phase chromatography and UV spectrophotometry, I examined whether the distribution of MAA compounds across different areas of the body is correlated with differential UV exposure. Comparisons were made between the MAA content and the absorbance spectra of mucus from the dorsal, ventral, caudal and head body surface areas in five species of Scaridae (Chlorurus sordidus, Scarus schlegeli, S. niger, S. psittacus and S. globiceps) from Ningaloo Reef, Coral Bay, Western Australia. All fish analysed had at least five MAAs present, and results showed that fish had increased UV absorbance in mucus over the dorsal area, which receives the brunt of UV radiation. Little UV protection was found in mucus from the ventral area, which receives the lower level of UV radiation mostly via reflection of the sand and reef surfaces. Furthermore, UV absorbance per mg dry mucus versus standard fish length showed that there is a positive relationship in C. sordidus with increasing size. I examined whether there is a difference in the quantity of UV screening compounds found in the mucus of fish along a longitudinal geographical gradient from inshore reefs (Lizard Island, Great Barrier Reef) to the outer edge reefs to oceanic reefs (Osprey Reef). MAA absorbance increased with longitudinal distance from the mainland landmass of Australia to more oligotrophic outer reefs, where UV attenuation is reduced and the ocean is more transparent to UV wavelength. I determined that fish living on inshore, more turbid reefs where UV attenuation in shallow waters is high have lower levels of MAA protection than fish from clear oceanic reefs. Furthermore, there seems to be a direct relationship between light attenuation and exposure with the quantity of protective sunscreening found in the mucus of reef fish. It is know that UV irradiation decreases with water depth and that mucus from fish with deep habitats absorbs less UV than that of fish from shallow habitats. It is unknown however, whether this UV protection is variable within the same individuals and if so, how fast changes 11 occur. To test this, I relocated 9 ambon damselfish from a deep reef (18 m) to a shallow reef (1.5 m) to expose fish to increased levels of UV and relocated another 7 fish from a shallow to a deep reef to expose fish to decreased levels of UV. One week after relocation, all fish were returned to their original reef site to determine whether MAA levels would return to their initial levels. Fish relocated to a shallower depth were recovered and had a 60% (SD+/-2%) increase in mucus UV absorbance. Conversely, the fish relocated to a deeper depth were recovered and had a 41% (SD+/-1%) decrease mucus UV absorbance. No difference was found between UV absorbance of relocated and original fish at both depth. Six days after fish were returned to their original reef, mucus UV absorbance levels had returned to 67% +/- 4% of the original level. These results show that mucus UV absorbance is variable in individual ambon damselfish and that the sunscreen protection typical for a certain depth is reached in relocated fish within just a few days of relocation. The rate of MAA loss is higher than the accumulation of MAAs suggesting that diet is not the sole determining factor involved in the sequestration of MAAs to mucus. The cleaner fish Labroides dimidiatus performs a mutualistic service by removing ectoparasites such as gnathiid isopods as well other dead infected tissue from its clients. Cleaner fish however are also known to feed on client mucus. The benefits of eating mucus until recently were unclear. In this study, we analysed the mucus of several cleaner fish clients to determine whether mucus feeding has a nutritional advantage over gnathiids and whether cleaner fish obtain their own MAA protection through this dietary mucus ingestion. Results show that host fish that are infected with gnathiids of poor nutritional value, in contrast to those that harbour gnathiids with higher nutritional value, continuously exude mucus that has both high nutritional value and high MAA content. These findings support the conclusion that in a competitive market for cleaners some host fish are forced to offer more than parasites to cleaners. Ultraviolet light that is not filtered by UV absorbing compounds such as MAA may still lead to DNA damage such as the formation of cyclobutane pyrimidine dimers (CPDs) or 6-4 photoproducts (6-4 PPs). However, coral reef fish have alternative mechanisms to overcome UV induced damage via the photolyase DNA repair mechanisms. We experimentally demonstrated for the first time that a coral reef fish species, the moon wrasse Thalassoma lunare has the ability to repair DNA damage via photoreactivation. Fish both with and without MAA protection were irradiated with UVB wavelength to induce DNA lesions. Half of the experimental fish were then exposed to photoreactivating wavelength to induce DNA repair 12 while the other fish were blocked from the repair mechanisms. Fish which had undergone DNA repair had the lowest number of lesions regardless of mucus MAA protection. When fish were blocked from photoreactivation wavelengths MAA sunscreens clearly served a photoprotective role. The amount of damage was greatest in fish which both lacked MAAs and which were also blocked from photoreactivating wavelengths. Thus for the overall UV protection of fish both the MAA sunscreens as well as the DNA repair system play a significant role in counteracting UV damage. Ultraviolet protection by MAA sunscreens is ubiquitous in marine fish. To date the same 5 MAA compounds (palythine (λmax 320 nm), asterina (λmax 330 nm), palythinol (λmax 332 nm), usujirene (λmax 357 nm) and palythene (λmax 360nm) have been identified in the mucus of several different species of reef fish from Australia. Here we report the first evidence of the presence of additional UV absorbing compounds found in the mucus of fish from Indonesia. Using UV spectroscopy the mucus of four species of fish was compared between both geographical regions. The presence of an additional peak between 294-296 nm wavelengths suggests the presence of gadusol and/or deoxygadusol, which are photoprotective compounds, thought to be the precursors of MAAs. Thus, UV protecting compounds in the mucus of fish may not be as conserved between different regions as previously assumed. Our knowledge concerning the effect of UV radiation has advanced considerably in the past decade and my research findings contribute to the better understanding of protective mechanisms of marine fish. The correlations I have found between UV attenuation/exposure, depth, and longitude of sampled individuals lead me to believe that mucus UV absorbing MAA compounds are a highly efficient adaptive defence.