The sustainable growth of the aquaculture industry will depend on novel strategies that work to enhance productivity and reduce impacts on the environment. This will be necessary to meet current and future demands for seafood. Optimising feeds in aquaculture is a primary route used to improve productivity and sustainability. Typically, aquafeed research focuses on altering the type, quantity or ratio of individual macronutrients. Throughout this thesis I present an alternative route of feed formulation that centres on understanding the integrative physiology of fishes and investigates the effect of diet on energy use. I show how this approach can be used as a tool to produce physiologically informed aquafeeds that aim to maximise feed efficiency and improve aquaculture sustainability. To begin, in chapter 1 I present a broad overview of the literature, ideas and theories discussed and examined throughout this thesis. I identify the current gaps in knowledge and detail the overall aim and scope of this work. In this chapter I discuss the potential energetic and physiological costs that could be associated with the non-nutritive components of a feed. Specifically, I consider the potential limitations associated with feeding on diets with high dietary buffering (ability to resist changes in pH) and highlight the capacity to exploit this feed characteristic to reduce the energetic and physiological cost of digestion to enhance fish growth. In chapter 2 I address this theory from the perspective of fish in the wild. Fish in the wild consume whole prey including hard skeletal parts like shell and bone. The calcium minerals that make up shell (CaCO3) and bone (Ca3(PO4)2) have buffering properties that are resistant to changes in acidity. Given this, it was expected that fish feeding on a diet supplemented with these calcium buffering minerals would have physiological consequences for acid secretion at the stomach, the associated blood alkaline tide (postprandial rise in blood pH and HCO3-), net-acid base excretion, the energetic cost of digestion and fish growth. When compared to juvenile rainbow trout (Oncorhynchus mykiss) fed a non-buffering CaCl2 control, fish fed diets supplemented with CaCO3 or Ca3(PO4)2 had a more alkaline stomach pH, a greater alkaline tide, net base excretion and total cost of digestion. Surprisingly the greater energetic and physiological burden placed on fish consuming a more buffered meal were not great enough to influence fish growth over a 14-day period. For the first time, this chapter confirmed the influence of dietary buffering on the energetic and physiological costs associated with digestion in fish. I discuss these results within the context of aquaculture sustainability and the ecology of fish in the wild, including the potential influence of dietary buffering on prey choice and trophic interactions both now and into the future. Building on this idea, in chapter 3 I worked to develop a physiologically informed acidified barramundi (Lates calcarifer) pellet. In contrast to chapter 2, it was expected that by reducing the dietary buffer capacity of a feed through dietary acidification, the physiological costs of acid secretion and acid-base regulation during feeding and digestion could be reduced. As expected, when fed an acidified diet barramundi had a more acidic stomach chyme pH, and a much-reduced alkaline tide and net base excretion to the external environment. For the first time I show the presence of an alkaline tide in this species, and detail how this response can be modulated by dietary buffering. However, this study did not determine whether this reduction in physiological costs translated to changes in energy use or fish growth. Therefore, in chapter 4 I assessed the effect of diet acidity on the energy cost of digestion and fish growth. Juvenile barramundi fed on an acidified diet had a reduction in the total cost of digestion by 45 %, which translated to an increase in feed efficiency by 14 % at day 14. Unexpectedly, observed improvements in growth efficiency declined over time so that feed efficiency was no longer significantly different between groups at day 28. An interesting result observed in chapter 2 and 4 was the influence of dietary buffering on the peak of oxygen consumption during digestion. Specifically, I observed that fish feeding on an acidified diet had a reduction in the peak of oxygen consumption by ~ 18 % when compared to fish feeding on a control diet. Therefore, in chapter 5 I investigated the potential of diet acidity to improve the thermal tolerance of fish. Under warming the SDA becomes temporally compressed, increasing peak oxygen consumption and reducing the post residual aerobic scope. As a result, during warming, fish in aquaculture will reduce feed intake which can compromise growth and make fish more susceptible to disease and/or mortality. To maximise PRAS and any possible energy savings during feeding, diet acidity was increased to represent the optimal acidity of the stomach during digestion in barramundi (pH 2 – 3). Given the observed influence of diet acidity on oxygen consumption during feeding from chapter 4 it was expected fish fed on the acidified diet would eat more and grow more efficiently at supraoptimal temperatures. In contrast to this prediction, barramundi exposed to elevated temperatures (37 ± 1 °C) and feeding on an acidified diet, ate less, grew less efficiently, and had increased mortality. The start pH of the experimental feed used in this study was pH 2.5, which was vastly more acidic than the start pH of 4.1 of the acidified diets used in chapters 3 and 4. It was therefore determined that the increased acid content used to produce this diet may have compromised some aspects of digestion to affect fish growth, performance, and survival. Despite this unexpected result, this study still worked to confirm that dietary acidification has a ‘tipping point’ and showcased the care that must be taken when choosing the type and quantity of acid used, the actual pH of the diet reached and the optimal acidity of the gut in the target species. The results observed in chapter 4 and 5 confirmed that chronic exposure to an acidified diet could be compromising certain aspects of acid-base regulation and creating a greater physiological burden that affects fish growth over time. Therefore, to better understand the long-term chronic effects of diet acidity on digestion and acid base regulation, in chapter 6, I used quantitative RT-PCR to investigate the expression of H+/K+ Atpase (atp4-α) in the stomach and Cl-/HCO3- anion exchangers (slc26a3) in the intestine of juvenile barramundi fed a control, acidified or HEPES-buffered diet after 25 days. As a result of reduced requirement for endogenous acid secretion, enhanced H+ intake, and therefore greater need for chyme neutralisation at the intestine, I expected that fish fed an acidified diet would experience a downregulation of atp4-α at the stomach and an upregulation of slc26a3 in the intestine when compared to the control. In chapter 6, I confirmed the presence of the protein coding genes atp4-α in the stomach and slc26a3 in the intestine. As expected, I observed a signficant 4.8-fold downregulation of atp4-α in the stomach of fish fed an acidified diet, and an almost 50-fold upregulation of the same stomach gene in fish fed a buffered diet. Unexpectedly, I observed a 3.8-fold down regulation of slc26a3 in the intestine. These results suggest that the expression of these genes in barramundi can be modulated by diet acidity, and that barramundi atp4-α synthesis is inhibited by luminal H+ and that the expression of slc26a3 is modulated by basolateral (i.e., blood) HCO3- concentration and the alkaline tide. This works goes futher to explain the observed differences in energy use of fish fed diets of varying buffer capacity, and highlights how enhancing diet acidity could work to aid some aspects of acid base regulation but impair others. Given these findings, I discuss how chronic exposure to dietary acidification could compromise chyme neutralisation at the intestine to influence fish growth over time. These results suggest that whilst gastric acidification might be made energetically easier and optimal gastric digestion might occur sooner after meal ingestion, these benefits are countered by the repercussions of an acidified diet for intestinal processes. Further research is required to determine if dietary acidification can be beneficial long term. Finally in chapter 7, I discuss the broader implications of this work including its general limitations, future directions, and applications to sustainable aquaculture. I use the results from this thesis to detail the importance of a holistic approach to feed formulation, sustainable aquaculture and the study of fish ecophysiology.