Respiratory and nutritional physiology of spiny lobster juveniles in culture

The spiny lobster Sagmariasus verreauxi is the largest species in the Palinuridae family and an important commercial seafood product in the Southern Hemisphere. The recent closure of the lifecycle of S. verreauxi from eggs to adult in captivity has improved the pathway to sustainable aquaculture for this species. The ability to culture an aquaculture species to harvest in a sustainable manner requires a plethora of nutritional and physiological knowledge. One piece of this puzzle is understanding the processes involved in energy metabolism. In particular, understanding nutritional physiology in terms of nutritional status will help develop effective nutritional regimes to optimize growth and achieve more sustainable aquaculture. The goal of this thesis was to investigate the nutritional physiology in cultured juvenile S. verreauxi of different nutritional status. In detail, this thesis consists of four linked but self-standing chapters addressing the following: the contribution of protein synthesis to energy metabolism using a protein synthesis inhibitor cycloheximide before being measured directly (Chapter 2), the potential use of a non-destructive stoichiometric approach to examine metabolic energy substrate (protein/amino acid, lipid or carbohydrate) use in aquatic ectotherms (Chapter 3), the use of the stoichiometric approach to examine metabolic energy substrate use and specific dynamic action (SDA) in lobsters fed a natural feed squid (Nototodarus sloanii) (Chapter 4), and the effects of different dietary protein on energy metabolism including protein synthesis (Chapter 5). Lobster metabolic rates declined with starvation and cycloheximide-sensitive protein synthesis in unfed lobsters represented a minor component of energy metabolism. In contrast, protein synthesis accounted for a major proportion of SDA in squid-fed lobsters. These results suggest the contribution of protein synthesis to energy metabolism in crustaceans varies with nutritional status, S. verreauxi SDA is predominantly a post-absorptive process mainly related to growth, and that high dietary digestible energy content is essential as growth-related protein synthesis is energetically expensive. Thereafter, to better understand energy metabolism in aquatic ectotherms, a review was provided to illustrate the use of the stoichiometric approach to examine metabolic energy substrate use. This approach is based on the evaluation of respiratory quotient (RQ) and nitrogen quotient (NQ), determined by simultaneous measurements of respiratory gas (O\\(_2\\) and CO\\(_2\\)) exchange and nitrogenous (ammonia and urea) excretion. This review is timely both because of technical advances in measuring total CO\\(_2\\) in seawater and the benefits that the stoichiometric approach provides for understanding energy metabolism. This approach enables repeated evaluations of major metabolic energy substrate use in the same animal at any one time, allowing the interpretation of bioenergetic change within a daily cycle. Protein was the primary energy substrate for 2-day fasted lobsters with lipid accounting for the remainder, and lipid became the main energy substrate after 10-day starvation, indicating lipid oxidation increased with the extent of fasting. Squid-fed lobsters predominantly oxidized protein to provide energy during SDA, and lipid and carbohydrate provided significant energy at different postprandial times. This suggests suitable amounts of high-quality protein with major non-protein energy-yielding macronutrients, lipid and carbohydrate, are oxidized and therefore should be included in lobster feeds. It is important to note that the stoichiometric approach is only feasible in conditions where the RQ is within the theoretical range (0.71-1). However, RQ exceeded 1 on a number of occasions in various aquatic ectotherms, including S. verreauxi in my study, indicating the need for additional studies to decipher the limitations and applicability of the stoichiometric approach. To investigate the effects of different dietary protein on energy metabolism, the measurement of SDA and metabolic energy substrate use using the stoichiometric approach was combined with the assessment of whole-body protein synthesis using a non-destructive endpoint stochastic model. Three isoenergetic feeds were formulated with varying crude protein: 40%, 50% and 60%, corresponding to CP\\(_{40}\\), CP\\(_{50}\\) and CP\\(_{60}\\) treatments, respectively Similar to the SDA magnitude in squid-fed lobsters estimated by the stoichiometric approach and traditional oxycalorific coefficient approaches, there was no difference in the SDA magnitude estimated by varying approaches in lobsters fed each formulated feed. Total CO\\(_2\\) and ammonia excretion, SDA magnitude and coefficient, and protein synthesis in the CP\\(_{60}\\) treatment were higher compared to the CP\\(_{40}\\) treatment. These differences demonstrate dietary protein influences post-prandial energy metabolism. The average contribution of protein oxidation was lowest in the CP\\(_{50}\\) treatment, suggesting the mechanisms underlying the most efficient retention of dietary protein and the optimum dietary inclusion. The combined information advances the understanding of how deficient and surplus dietary protein affects energy metabolism and provides an approach for the fine-scale evaluation to support sustainable aquaculture. Overall, this thesis provides the first stoichiometric investigation of the metabolic energy substrate use and SDA in a spiny lobster species, by successfully measuring carbon dioxide excretion combined with simultaneous measurement of oxygen consumption and nitrogenous excretion. In addition, this thesis provides the first examination of the contribution of protein synthesis to decapod energy metabolism by using a protein synthesis inhibitor. Furthermore, the thesis determined whole-body protein synthesis using an endpoint stochastic model, and explored the relationships between protein synthesis, SDA and energy substrate oxidation. The combined measurements provide detailed nutritional and physiological information relevant to crustacean growth and have applications for the evaluation of feeds and feeding regimes. In future, it would be beneficial to investigate the use of the stoichiometric bioenergetic approach in other emerging aquaculture species and expand applications of the endpoint stochastic model when examining the effect of dietary protein quality on protein metabolism.