The isolation, identification and exploration of the biophysiological significance of plasma biliverdin in the ballan wrasse (Labrus bergylta)

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.