Morphological and functional modifications of the gastrointestinal tract during metamorphosis in Atlantic halibut (Hippoglossus hippoglossus)

Metamorphosis in flatfish is the transition from a symmetrical larva to an asymmetrical juvenile stage, allowing the organism to utilize new resources in a different habitat and thus providing an evolutionary advantage. In addition to the overt changes in external morphology, coordinated maturation of many tissues and organs occur, including the gastrointestinal (GI-tract). In flatfish species, the GI-tract is extensively remodelled during larval development and its appropriate development is crucial for adapting to the shift in habitat and diet that accompanies the transition into a juvenile. Thyroid hormones (THs) play a central role in this process, translating the environmental cues into a coordinated program that remodels the organism. This thesis uses Atlantic halibut (Hippoglossus hippoglossus), a valuable aquaculture species, to provide new insights into flatfish metamorphosis. The transition from larvae to juvenile is here explored based on transcriptional changes during metamorphosis, functional development of the GI-tract and the establishment of neuroendocrine pathways for appetite-regulation. To study the main molecular mechanisms associated with Atlantic halibut metamorphosis, reference transcriptomes of the GI-tract, head and skin were generated using 454 pyrosequencing. The resulting large set of good quality reads has been assembled into a significant number of contigs and successfully annotated using a multi Blast step approach. Functional analyses revealed that the most prominent biological processes are equally common between the three regions analysed (GI-tract, head and skin) despite significant differences in tissue complexity. Additionally, unique sets of biological processes associated with tissue-specific morphology and function were identified for each region. For the GI-tract, the focal organ of the present thesis, a total of 206 GO-terms were found to be unique, including gastric acid secretion process. Using SOLiD sequencing technology, it was revealed that during Atlantic halibut development hundreds of genes are significantly (p<0.05) up- or down-regulated at the whole larvae level, indicating that many key transcriptional modifications underlie the significant changes in tissue that occur between premetamorphic and juvenile stages. It is well established that initiation of metamorphosis in Atlantic halibut is associated with a surge of THs, thus THs levels increase until the metamorphic climax and decrease in the post-climax stage. The TH cycle during metamorphosis was accompanied by a change in the expression profile of key elements involved in the thyroid signalling pathway, and the change in their expression profile occurred in synchrony with increasing THs levels at metamorphic climax. The coordinated changes in gene expression of TH signalling pathway players, THs levels and tissue morphology confirm the importance of the TH system in orchestrating the Atlantic halibut postembryonic development. The list of candidate genes for future studies aimed at understanding GI-tract development during metamorphosis includes many TH responsive genes. The functional ontogeny of the GI-tract was studied using an integrative approach to test whether the multiple functions of the Atlantic halibut stomach develop synchronously during metamorphosis. The onset of gastric function was determined using in vivo pH analysis in the GI-tract lumen, combined with quantitative PCR (qPCR) of the gastric proton pump α and β subunits and pepsinogen A2. The results indicate that gastric function is established during metamorphic climax. A 3D model series of the GI-tract development and in vivo observations imply that the stomach’s short-term reservoir function is established before metamorphosis, although the midgut acts as the main storage compartment until this function shifts to the stomach as its volume increases at metamorphic climax. The motility function of the GI-tract was investigated using in vivo analysis and results show that phasic and propagating contractions are established well before metamorphosis, but the number of contractions registered in the midgut decreases synchronously with the stomach’s increasing peristaltic activity at metamorphic climax. The stomach’s role in appetite control was studied via changes in ghrelin mRNA expression. Ghrelin is mainly produced in the stomach and known as an important orexigenic factor in mammals and some fish species. During metamorphosis climax of Atlantic halibut, ghrelin levels significantly (p<0.05) increased parallel to stomach development. However, ghrelin expression did not change in developing Atlantic halibut in response to food intake. To explore potential changes in appetite regulation in Atlantic halibut during metamorphosis, spatial and temporal gene expression patterns of neuropeptide Y (NPY), peptide YY (PYY), pro-opiomelanocortin (POMC-C) and cocaine- and amphetamine-related transcript (CART) in the brain were analysed. NPY, PYY and POMC-C gene expression did not change during ontogeny, but CART was significantly (p<0.05) down-regulated when metamorphosis commenced. Only PYY gene expression responded significantly (p<0.05) to food intake in the premetamorphic stage. Results for the other appetite-regulating factors were inconclusive, however Atlantic halibut larvae fasted for 44 h fasting showed significant responses of PYY, NPY, POMC-C and ghrelin after refeeding (p<0.05). In summary, the results in this thesis reveal that the Atlantic halibut transcriptome is highly dynamic through post-embryonic development and that the TH signalling pathway is critical for the radical remodelling of organs during flatfish metamorphosis. The remodelling of the GI-tract, specifically the stomach development and volume growth, is linked to the surge of TH levels during the climax of metamorphosis, and the morphological modifications are connected with a set of functional changes. Furthermore, the development of the stomach during metamorphosis and emergence of proteolytic activity are correlated with a significant rise in stomach ghrelin, crucial for the gastric involvement in appetite regulation. Functional specializations of the GI-tract, such as its putative osmoregulatory function, are already well established at the onset of exogenous feeding and are therefore independent of metamorphosis. For the first time analysed, the neuroendocrine control of appetite regulation during Atlantic halibut larval development revealed no link between the onset of appetite-regulation with THs and metamorphosis, with the exception of ghrelin. Therefore, more studies will be required to better understand feeding and the onset of appetite regulation in fish larvae. Overall, the results in this thesis have opened up several new avenues of research and new molecular resources have been developed which will contribute to future studies aimed at understanding the complex process of flatfish metamorphosis