Application of a stoichiometric bioenergetic approach and whole-body protein synthesis to the nutritional assessment of juvenile Thenus australiensis

The present study successfully combined a stoichiometric bioenergetic approach with an endpoint stochastic model to simultaneously determine specific dynamic action, metabolic substrate use and whole-body protein synthesis in juvenile slipper lobster Thenus australiensis. Juvenile lobsters were fasted for 48 h to investigate routine metabolism before receiving a single meal of formulated feed containing 1% 15N-labeled Spirulina. Postprandial oxygen consumption rate, dissolved inorganic carbon, and total nitrogen excretion returned to the pre-feeding level within 24 h. The rate of whole-body protein synthesis was 0.76 ± 0.15 mg CP g−1 day−1, with a significant reduction from 24 to 48 h post-feeding. The postprandial increase in whole-body protein synthesis accounted for 13–19% of total oxygen uptake. Protein was the primary energy substrate for 48 h fasted (45% oxygen consumption) and post-feeding lobster (44%), suggesting that dietary protein was not efficiently used for growth. The secondary energy substrate differed between carbohydrates in 48 h fasted and lipids in post-feeding lobsters. The present study recommends integrating protein synthesis into protein requirement experiments of marine ectotherms to acquire a more comprehensive picture of protein and energy metabolism and nutritional physiology crucial for formulating cost-effective aquafeeds.

Experimental animals.Juvenile T. australiensis were hatchery-reared from egg by a commercial lobster hatchery (Ornatas, Toomulla Beach, Australia).After arrival, lobsters were maintained in experimental aquaria (0.38 m length × 0.24 m width × 0.25 m height, 18 L) at the Institute for Marine and Antarctic Studies (IMAS), Hobart, Australia.Table 1.Experimental feed formulation and chemical composition on a dry matter basis.Experimental feeds were subsampled (n = 2 per feed) to reflect the average feed composition over the experimental phase.A Commercial in confidence basal mix.B Spirulina powder, paddymelon, The Melbourne Food Depot.C Algal lyophilised cells-15 N (Spirulina), 98 atom % 15 N, Sigma-Aldrich.D NFE (Nitrogen free extract) = 100 − (crude protein + total lipid + ash).E Calculated by using factors 23.9 MJ kg −1 , 39.8 MJ kg −1 and 17.6 MJ kg −1 for proteins, lipids and carbohydrates (NFE), respectively 27 .Apparent digestibility of Spirulina.A total of 57 juvenile lobsters with an average (± S.E.) initial wet weight of 26.0 ± 1.4 g were randomly allocated to 9 experimental tanks (038 m length × 0.24 m width × 0.25 m height, 18 L).Three tanks were randomly assigned to each of the three experimental feed treatments.Tanks were supplied with filtered, ozonated seawater at a rate of six exchanges h −1 and maintained under a 12:12 h blue light: dark photoperiod, set up as a flow-through culture system.Dissolved oxygen (104.2 ± 0.7% saturation), salinity (33.8 ± 0.0 ppt), pH (8.2 ± 0.0) and temperature (26.6 ± 0.7 °C) were recorded daily to ensure high water quality.Experimental feeds were supplied in excess of requirements continuously over 18 h d −1 (approximately 15:00 to 09:00 h daily) using belt feeders, with a daily ration of approximately 2% of body weight on a wet weight: wet weight basis.Belt feeders were loaded daily with a pre-weighed ration of feed derived from each tank's initial total lobster biomass.The feed ration was adjusted through the assessment of mortalities.Uneaten feed was cleaned by siphon.Mortalities and moulting events were recorded daily, and exuviae were removed from the tanks as soon as they were observed to prevent animals from feeding on them.Apparent digestibility was measured as described in Wirtz, et al. 2 .Briefly, after six days of acclimatisation to the assigned experimental feeds, faeces were collected daily from each tub following the first feeding at 15:00.Faeces were collected with a disposable pipette onto a 250 µm screen and rinsed with distilled water to remove the salt.Daily faecal collection from a single tank was pooled over 48 days and stored at − 20 °C until chemical analyses.The apparent digestibility coefficient (ADC) of the reference and the experimental feeds was calculated according to 2,28 : where ADC DM represents the ADC of dry matter (DM) in the feed; Y Feed and Y Faeces signify the proportion of the inert marker yttrium oxide in the feed and faeces, respectively; ADC N represents the ADC of nutrients crude protein (CP), total lipid (TL) and gross energy (GE); N Feed and N Faeces are proportion (%) of CP, TL and GE in the feed and faeces, respectively.

Ingredient
The apparent digestibility of Spirulina was calculated based on the 70:30 and 85:15 ratio of reference feed to test feed 29 : where AD Ing % = AD of nutrients in Spirulina; AD TF = AD of nutrients in the test feed; AD RF = AD of reference feed; P RF = basal mix proportion; P Ing = Spirulina proportion; N RF = nutrient concentration in reference feed and N Ing = nutrient concentration in Spirulina.
Oxygen consumption and protein synthesis.Experimental setup.After the digestibility experiment, the remaining 38 lobsters were transferred from the experimental tanks into individual cylindrical vessels (3 L) for holding during acclimatisation to constant dim light to avoid interference from circadian rhythms 17 .During acclimatisation, lobsters were fed ad libitum with the reference feed of the digestibility experiment but formulated with 1% unlabelled Spirulina.After four weeks of acclimatisation, lobsters were closely monitored for moulting events.Five lobsters with an average weight of 53 ± 2 g wet weight (WW, 9.62 ± 0.32 g dry weight (DW)) were used for the experiment.As soon as lobsters showed a constant feed intake (about 1% of their body weight) post-moult, they were weighed and transferred to the cylindrical experimental vessels (3 L) that were equipped with a submersible aquarium pump (AP210 Water feature pump, AQUAPRO, Forrestdale, Australia), luminescent dissolved oxygen optode (Hach LDO, HQ40d, Hach Company, USA) and an air stone connected to the central air supply.The seawater level was maintained at 2.8 L + body volume.Lobsters were fasted for 48 h, ensuring the same post-absorptive status.
Oxygen consumption rate and SDA.The oxygen consumption rate (ṀO 2 , mg O 2 g DW −1 h −1 ) was determined according to Wang, et al. 6 .During the last 2 h of the 48 h fasting period, the routine metabolic rate (RMR, mg O 2 g −1 h −1 ) of each lobster was determined (from 06:00) based on the measurement of ṀO 2 30,31   .Briefly, ṀO 2 was calculated from the decline in dissolved oxygen concentrations in the experimental vessel during the experiment measured with a luminescent dissolved oxygen optode (Hach LDO, HQ40d, Hach Company, USA).Experimental vessels were covered with a blue transparent solar pool cover (Intex Development Co., Ltd., Hong Kong) to limit oxygen diffusion with the environment.Aeration and seawater flow were manually halted for a 20 min ṀO 2 measurement period, followed by a 10 min re-oxygenation period.The halt-restart process was repeated three times, and the RMR was determined as the mean of the three ṀO 2 measurements, where the background ṀO 2 for each lobster was subtracted [32][33][34] .Background ṀO 2 was determined 2 h before stocking the animal by the same process but in an experimental vessel without a lobster.
The RMR measurements were followed by a 1.5 h feeding period.Lobsters were fed with 15 N labelled experimental feed at a ration of 1.5% of their body weight (average body weight 0.71 ± 0.03 g DW).Subsequently, uneaten feed was collected for apparent feed intake (AFI) determination.The seawater level in the experimental vessel was dropped to 1.3 L + body volume by siphoning 1.5 L of seawater into a 5 L plastic flask via Tygon E-3603 tubing (Saint-Gobain Performance Plastics, Charny, France).After that, 1.5 L of freshly filtered seawater was added into the experimental vessel, and the lobster was subjected to the 20 min seawater addition and ṀO 2 measurement protocol (as described above) at 2 h intervals for the first 12 h, followed by a 24 h and 48 h to provide nine postprandial ṀO 2 measurements for the determination of SDA.Six SDA variables were individually identified: (1) SDA peak , determined as the peak post-feeding ṀO 2 (mg O 2 g DW −1 h −1 ); (2) time www.nature.com/scientificreports/ to SDA peak , determined as the time from feeding to SDA peak (h); (3) SDA duration, determined as the duration that the average post-feeding ṀO 2 remained greater than the RMR (h); (4) SDA magnitude (TṀO 2 , mg O 2 g DW −1 ), calculated by the total increase in ṀO 2 above the RMR; (5) E SDA (J g −1 ) was determined using a simplified traditional approach where SDA magnitude was converted to energy using an empirical oxycalorific coefficient (Q OX ) of 13.84 J mg −135 and using a stoichiometric bioenergetic approach 17 , where E SDA = 11 × TṀO 2 (mg O 2 g DW −1 ) + 2.6 × TMCO 2 (mg g DW −1 ) − 9.5 × TMTAN (mg g DW −1 ) − 2.44 × TMurea (mg g DW −1 ), representing the accumulated CO 2 , NH 3 and urea excretion during SDA, respectively; (6) SDA coefficient (C SDA , %) calculated by dividing E SDA by the energy in the ingested feed (J g −1 ).
Seawater sampling.During the RMR determination, a 20 mL seawater sample was collected within 3 s at the start and end of each ṀO 2 measuring period via a 20 mL syringe (Terumo Co., Ltd., Japan) to determine routine excretion rates of total dissolved inorganic carbon (MDIC, μmol g DW −1 h −1 ), total ammonia-N (MTAN, mg g DW −1 h −1 ) and urea-N (Murea-N, µmol g DW −1 h −1 ), after correction for background levels.The methods used for seawater sampling were based on Wang, et al. 17 .In brief, 14 mL seawater from the collected 20 mL was dispensed into a 12 mL precooled glass vial (Labco Limited, Lampeter, UK) until overflow to minimise air-water gas exchange and disinfected with 3.6 μL of saturated mercuric chloride.The dispensation and disinfection were completed within 1 min.The vial was subsequently capped and stored at 4 °C until MDIC measurement.The remaining 6 mL were used to determine nitrogenous excretion, with 3 mL sealed in a 10 mL disposable plastic vial for MTAN measurement, disinfected with 10% chloroform to prevent bacterial activity and the remaining 3 mL sealed in another 10 mL vial for Murea-N measurements.Seawater samples for MTAN and Murea-N measurement were stored at − 20 °C within 20 min after sampling and thawed at room temperature before analysis.
After feeding, the siphon-collected 1.5 L seawater sample was immediately acidified with 3.75 mL of 4 M HCl and transferred and stored at 4 °C in a 5 L round bottom glass flask (Schott Duran, Mainz, Germany) for the determination of the initial 15 N concentration 36,37 .Concurrent with postprandial ṀO 2 measurements, 20 mL seawater samples were collected at the start and end of each 20-min seawater and airflow halt cycle to determine the postprandial MDIC, MTAN and Murea-N.At 12, 24 and 48 h post-feeding, 1.5 L of seawater were collected and stored for the later determination of 15 N ammonia enrichment 38,39 .After each measurement, the extracted water was replaced with freshly filtered seawater.After adding fresh seawater for the last time at 48 h, the lobster was euthanised in ice seawater slurry, weighed, measured, and dissected to remove the whole hepatopancreas (HP) and approximately 1 g WW of tail muscle tissue (TM).The carcass remains, HP and TM samples were individually stored at − 20 °C in preparation for freeze-drying and chemical composition analysis.Followed by the dissection, the ṀO 2 , MDIC, MTAN and Murea-N in the experimental vessel were measured 3 times for 20 min to identify differences in background parameters before and after each experiment.
Nitrogen excretion analysis.Nitrogenous concentrations were measured colourimetrically using a Synergy HT Multi-detection Microplate Reader (BioTek Instruments, Winooski, VT, USA).The concentration of MTAN was determined by the salicylate-hypochlorite method 40,41 .The concentration of Murea-N was determined with the diacetyl monoxime method 42 , which was modified to increase sensitivity 43 .The correlation coefficients (R 2 ) of the linear calibration curves in MTAN and Murea-N determination were higher than 0.99.The duration (h) of MTAN, Murea-N and total nitrogenous (MTN, the sum of MTAN and Murea-N) excretion was determined when postprandial excretion rates returned to routine levels.
Instantaneous metabolic energy substrate use calculation.The instantaneous metabolic energy substrate use was based on Wang, et al. 17 .The fraction of aerobic energy substrate use supplied by protein (amino acid) (P), lipid (L) and carbohydrate (C) was calculated as follows: where 0.27 is the theoretical maximum nitrogen quotient (NQ) when protein (amino acid) is the only substrate being completely oxidised under aerobic conditions; m is the aerobic respiratory quotient (RQ) for protein (amino acid) oxidation, determined by 0.96 × MTAN% + 0.83 × Murea-N%, where 0.96 and 0.83 are the aerobic RQ for protein (amino acid) oxidation when ammonia and urea are the unique nitrogenous end-products, respectively; MTAN% and Murea-N% represent the contribution of MTAN and Murea-N to MTN (total nitrogenous excretion), respectively.The NQ was calculated as MTN/ṀO 2 and RQ as MDIC/ṀO 2 , where MTN, ṀO 2 and MDIC were expressed as µmol g DW -1 h -1 ("Nitrogen quotient and respiratory quotient calculation").
Protein synthesis.Ammonia from the 1.5 L collected seawater was distilled into boric acid to form ammonium borate.Full details of ammonia distillation are given by Carter, et al. 39 with modifications.The acidified sample P = NQ/0.27N enrichment (expressed as atom per cent excess, APE) of ammonia.Whole-body fractional protein synthesis rates (proportion of protein mass synthesised per day as a percentage, k s , % day −1 ) were calculated based on the 15 N enrichment of ammonia, using the endpoint stochastic model in conjunction with the determined wholebody protein content (protein pool) 36,39 .Protein consumption rates (k c , % day −1 ) were calculated based on the final protein content of the lobster (g DW protein consumed g DW lobster protein −1 day −1 , Houlihan, et al. 18 ).Anabolic stimulation efficiency = 100 × k s /k c ) 13 provided further analysis of protein utilisation.Protein retention rates (k g ) were calculated as the difference between digestible protein intake (dietary amino acid) and nitrogen excretion 44 .Protein degradation rates (k d ) was calculated from k g and k s measurements following the equation k Two conversion factors were used to convert absolute protein synthesis rates into equivalent oxygen consumption values, the minimum theoretical cost of 46 mmol ATP g protein synthesis −1 and a determined energy cost of 69 mmol ATP g protein synthesis −120 , assuming that 5.24 mg O 2 mmol ATP −113 .

Chemical composition analyses.
All samples, including experimental feeds, protein sources and animals, were freeze-dried (FD) to a constant weight and subsequently prepared for biochemical analysis by first grinding to a homogenous powder mechanically using an analytical mill (A11 basic Analytical mill, IKA®) and then manually using a mortar and pestle.Dry matter (DM) of FD samples was determined gravimetrically after oven drying at 105 °C for 24 h 45 .All biochemical analyses were performed on FD samples and corrected for DM.Ash content was determined by the combustion of FD samples in a furnace at 600 °C for 2 h 46 .Crude protein content was determined after measuring the elemental nitrogen (N) composition of FD samples using a flash combustion isotope ratio mass spectrometry (varioPYRO cube coupled to isoprime 100 mass spectrometer) 47 .A conversion factor of 6.25 × N was used to calculate crude protein.The total lipid content was determined gravimetrically using a modified method from Bligh and Dyer 48 .Briefly, total lipid was extracted in a mixture of dichloromethane, methanol and milliQ water (1:1:0.9v/v/v) Yagiz, et al. 49 , except for the substitution of chloroform by dichloromethane 50 .Nitrogen free extract (NFE) was calculated as; NFE = 100 -(crude protein + total lipid + ash), g kg −1 DM basis.Energy content was calculated using factors 23.9 MJ kg −1 , 39.8 MJ kg −1 and 17.6 MJ kg −1 for proteins, lipids and carbohydrates (NFE), respectively 27 .

Statistical analyses.
Mean values of the replicate aquarium (n = 3) in the digestibility experiment and the individual lobster (n = 5) for the protein synthesis determination are reported ± standard error of the mean (S.E.).All statistical analyses were performed using RStudio (Version 1.2.5042, © 2009-2020 RStudio, Inc.).Before analysis, all percentage data were arcsine-transformed.Data were tested for normal distribution and homogeneity using the Shapiro-Wilk and Bartlett's test, respectively.Apparent digestibility coefficient of the experimental feeds, survival, instantaneous metabolic energy use and WBPS were analised by an ANOVA followed by a Tukey's HSD post hoc test.Different Spirulina levels and the energetic cost of SDA were tested with the help of paird student's t-test.A repeated measure of ANOVA was used to compare nitrogen quotient and respiratory quotient at different time points.All statistical tests were considered significant at p < 0.05.The magnitudes and duration of SDA were determined for each lobster individually (n = 5) by fitting fifth-degree (ṀO 2 and MDIC) and sixth-degree (MTAN and Murea) polynomial regressions to the experimental data.Magnitudes were calculated from the integral between the regression line and the routine metabolism using the function integrate() in R. Durations were determined by calculating the intercepts of the regression lines with the routine metabolism.
Instantaneous metabolic energy use.Protein (amino acid, 45%) was the predominant metabolic energy substrate in 48 h fasted lobster, followed by carbohydrate (35%) and lipid (20%, Fig. 7).The fractional contribution to metabolic use of protein (amino acid), carbohydrate and lipid fluctuated during the 48 h post-feeding, totalling 44%, 21% and 35%, respectively.The pairwise comparison revealed that protein (amino acid) had a significantly higher percentage of metabolic energy substrate use than carbohydrates (Tukey's HSD, p = 0.002), with no significant differences found between protein (amino acid) and carbohydrate with lipid (p > 0.05).Table 3. Apparent digestibility (AD, %) of dry matter (AD DM ), crude protein (AD CP ), and gross energy (AD GE ) of Spirulina using 85:15 (SP1) and 70:30 (SP2) inclusion levels for juvenile Thenus australiensis (mean ± S.E., n = 3).www.nature.com/scientificreports/Protein synthesis.The cumulative 15 N-labeled ammonia excretion rate (ce*) was measured over 48 h following a single feed (Fig. 8).The relationship between ce* and time after feeding was described as a negative exponential regression, showing a plateau between 24 and 48 h post-feeding.Thus, WBPS was estimated for 24 and 48 h following feeding using data collected over the first 48 h post-feeding.The whole-body protein content (protein pool) of juvenile T. australiensis was 503 ± 30 g CP kg −1 .The WBPS and fractional protein synthesis (Fig. 9 and Table 4) were determined to be 0.76 ± 0.15 mg CP g −1 day −1 and 0.15 ± 0.03% day −1 , respectively.Protein synthesis on the second day was significantly reduced (ANOVA, F 1 = 8.47, p = 0.020, Fig. 9) compared to day 1.The postprandial increase in WBPS accounted for 13 ± 3% and 19 ± 5% of total oxygen uptake, using the minimal and experimentally determined cost of protein synthesis in ATP g −1 protein, respectively.

Discussion
The present study validates the successful combination of a stoichiometric bioenergetic approach with an endpoint stochastic model for the simultaneous determination of SDA, metabolic substrate use and WBPS in aquatic ectotherms first used by Wang, et al. 6 .A comprehensive understanding of the contribution of protein synthesis to metabolism is key to investigating the relationship between nutritional status and feed nutritional quality and the effects on growth potential in crustaceans 13,15 .Thus, the present study provides comprehensive data on the major components of whole-animal metabolism, including respiratory gas exchange, nitrogenous excretion,  www.nature.com/scientificreports/specific dynamic action, metabolic energy substrate use, and whole-body protein synthesis in juvenile slipper lobster Thenus australiensis.Each of these components must be assessed individually and validated against previous research before they can be considered collectively to describe the protein-nitrogen flux revealing the nutritional status of the animal.The in vivo method for digestibility determination is subjected to different factors affecting its accuracy 51 .In juvenile white shrimp Litopenaeus vannamei apparent digestibility of test ingredients were significantly affected by the ingredient type and inclusion level, except for AD CP 51 .In the present study, spirulina levels resulted in significantly different apparent digestibility coefficients for the experimental feeds.However, Spirulina digestibility was not affected by different inclusion levels, and AD CP equalled 78 ± 3%.The AD CP for Spirulina, in the present study, was higher than previous reports of 52.6% in Macrobrachium tenellum 52 and 52.9% in S. verreauxi 6 .Since the same Spirulina protein was used for S. verreauxi 6 and T. australiensis, the different protein digestibilities may result from differences in the digestive capacity of these species, emphasising the importance of determining digestibility when protein synthesis is determined by 15 N-labeled nitrogen 36 .
SDA is a well-known metabolic event 16,53,54 , and following a meal, the flux of amino acids and other nutrients from the digestive system into other tissues stimulates a rapid increase in metabolism reflected by peaks in oxygen consumption, nitrogen excretion and protein synthesis 53,55 .Hence, postprandial oxygen consumption increases above the pre-feeding rate, often to a peak level, and then falls back to the pre-feeding level 54 .In the present study, oxygen consumption of T. australiensis agrees and showed a 2.3-fold increase in ṀO 2 directly after the meal, followed by a decline back to RMR within 24 h.
The energetic cost of SDA (E SDA ) estimated by two different approaches (one simplified traditional approach and one stoichiometric bioenergetic approach) were not significantly different and in agreement with previous findings for S. verreauxi 6 .However, since the ESDA values determined in the present study showed a difference of 14% (47 − 33%), further research is needed to understand if the traditional approach, using an empirical oxycalorific coefficient to convert SDA magnitude to energy, provides accurate E SDA results.The E SDA in the present study was notably higher than for other aquatic ectotherms 6,31 .At a ration of 1.5% BW, E SDA ranged from 3.0 J g −1 in kelp crab (Pugettia producta) to 10.9 J g −1 in purple shore crabs (Hemigrapsus nudus), and with increasing E SDA with increasing feed ration 31 .The SDA magnitude and, consequently, E SDA in aquatic ectotherms depends on body weight, feed composition, feeding ration, and temperature 16,31,54,56 .Hence, the SDA coefficient (C SDA ), calculated by dividing E SDA by the dietary energy content 54,57 , allows for intraspecific and interspecific comparisons of SDA that are independent of meal size, meal type, body size, and body temperature 31,53 .Further, the combination of C SDA and nitrogenous excretion provides information about dietary protein utilisation.Whereas optimal dietary protein can result in lower SDA and nitrogenous excretion 58 , an increase in C SDA and nitrogen excretion indicates increased protein metabolism and decreased efficiency in converting ingested nutrients to growth 6,56 .In the present study, the low C SDA of 2.31% and 1.62%, simplified traditional and stoichiometric bioenergetic approach, respectively, are comparable with previously reported C SDA for S. verreauxi (1.8-2.6%) 6, indicating efficient use of the provided formulated feeds.
As protein synthesis is a major contributor to SDA, it typically responds in a similar way, increasing after feeding before gradually returning to pre-feeding levels 15,16,20 .The present study results agree and show a parallel postprandial increase of ṀO 2 and k s , a return to RMR and a significant decrease of k s after 24 h.A significant proportion of SDA can be accounted for by the elevation of protein synthesis because SDA largely represents the cost of protein anabolism and hence growth 59 .The cost of WBPS in T. australiensis accounted for 13-19% (26 °C) of total oxygen uptake, agreeing with previous findings in crustaceans and fish 13,20 .
Ammonia-N excretion in juvenile T. australiensis was comparable with other decapod crustaceans 30,60 and represented the primary nitrogenous end-product with 83% of the total nitrogenous excretion.The Murea-N measured in the present study equalled 17% of the total nitrogenous excretion, similar to previous reports for Orconectes rusticus 61 .
Investigating the instantaneous metabolic substrate use provides a deeper understanding of how aquatic ectotherms oxidise energy substrates to provide energy and evaluate the dietary protein-sparing effect 17,23 .In the present study, the primary energy substrate oxidised in 48 h fasted lobster was protein (amino acid), followed by carbohydrate and lipid agreeing with other short-term fasted carnivorous decapods findings 17,62,63 .Provided protein (amino acid), carbohydrate and lipid were all oxidised at different proportions at different times to provide energy over 48 h post-feeding, indicating that besides protein, appropriate proportions of non-protein energy substrates, lipid and carbohydrate, are both likely to be essential for the formulation of cost-effective aquafeeds 17,64 .Table 4. Protein turnover and efficiency parameters for juvenile Thenus australiensis at 25.5 ± 0.3 °C (n = 5).k c protein intake, and k a digestible protein intake, k s protein synthesis, ASE Anabolic stimulation efficiency (k s /k c ). www.nature.com/scientificreports/Protein synthesis is central to growth; consequently, the study of WBPS is key to understanding the daily protein-nitrogen flux in aquatic ectotherms, which is crucial for optimising dietary protein in aquafeeds 6,24,25 .The present study used an endpoint stochastic model to calculate protein synthesis rates, previously applied to S. verreauxi 6 .Successful application of the single endpoint model depends on the 15 N-labeled nitrogen appearing in a single major nitrogenous end-product 36,37 .In the present study, ammonia was the major nitrogenous endproduct with low Murea-N excretion.Consequently, using 15 N-labeled ammonia to calculate WBPS is suitable for T. australiensis.The validation data on cumulative 15 N-labeled ammonia excretion rates showed a plateau within 48 h post-feeding, agreeing with findings for other aquatic ectotherms 65,66 .
Information on postprandial WBPS in crustaceans is limited 6,20,22,67 .Whilst in G. antarcticus (0 °C, 33 g) and isopod Saduria entomon (1 g, 4 and 13 °C) fed at 5% BW k s equalled 0.4% day −1 , 1.5% day −1 (4 °C) and 2.6% day −1 (13 °C), at the SDA peak , respectively 22,67 .Feeding (3% BW) C. maenas (15-18 °C, 54 g) showed a k s of 2.6% day −120 .The differences in postprandial k s can be explained by the combination of factors, including water temperatures, body weights, feeding regimes, methods and the different species used 15,67,68 .Using the same methodology, feeding regime, and dietary protein content of 60%, S. verreauxi (21 °C, 993 ± 23 g) showed a similar k s of 0.19% day −16 to the k s reported in the present study (0.15 ± 0.03% day −1 , 26 °C, 9.6 ± 0.3 g).However, since protein turnover is affected by BW and protein synthesis and protein degradation decline with increasing BW 11,69 , T. australiensis, in the present study, should have presented higher protein synthesis rates than S. verreauxi.A major driver of metabolism is the nutritional status of the animal, governed by feeding, nutrient intake and quality, and time without food 15,33 .Thus, low k s may be due to the poor nutritional status of the lobsters, resulting in low feed intake, which was lower than previously reported for juvenile T. australiensis 8 .
The comprehensive investigation on the major components of whole-animal metabolism contributes to our understanding of nutritional status and feed nutritional quality in relation to WBPS.Measuring protein synthesis and protein-nitrogen flux provides a subtle approach to assessing the efficiency of dietary protein utilisation 13 , thereby expanding knowledge of nutritional physiology and energy metabolism.Protein-nitrogen flux describes the partitioning of consumed and absorbed protein-nitrogen into protein syntheses and growth.In a simple nitrogen budget, the daily protein-nitrogen flux derived from consumed protein partitioned between faecal waste (faecal nitrogen), metabolic waste (total nitrogen excretion), and growth (protein retention) 13,70,71 .Fractional protein synthesis rates from the present study (Table 4) were used to model the protein turnover rates of a theoretical 50 g WW juvenile T. australiensis calculated for the first 24 h following consumption of a single meal based on Carter, et al. 44 .The protein-nitrogen flux was separately calculated for lobster with a positive (n = 2, Fig. 10a) and negative protein retention (n = 3, Fig. 10b).Increasing feed intake and corresponding digestible protein intake (dietary amino acids) increased protein synthesis and protein retention, agreeing with previous findings for the grass carp (Ctenopharyngodon idella) 44 .Since all lobsters were fed the same experimental feed, the protein loss can be explained by the poor nutritional status of the lobsters, displayed by low protein consumption and protein synthesis 15,33 ..

Conclusion
The present study successfully combined a stoichiometric bioenergetic approach with an endpoint stochastic model for the simultaneous determination of SDA, metabolic substrate use and WBPS in juvenile T. australiensis.The combined approach has great potential to provide crucial nutritional and metabolic information about the nutritional status of T. australiensis.The low k s determined in the present study can be linked to the poor nutritional status of the animal governed by low feed intake.A balanced feed containing suitable protein and other non-protein energy substrates such as lipid and carbohydrate is essential to spare dietary protein and increase protein efficiency for growth.In the present study, protein was the primary energy substrate, suggesting that dietary protein was not efficiently used for growth.Consequently, more research is required to investigate the effect of dietary protein on routine and postprandial nutritional physiology and bioenergetics.Thus, future research should consider integrating protein synthesis into protein requirement experiments of marine ectotherms to acquire a more comprehensive picture of nutritional requirements and efficiency.

Figure 1 .
Figure 1.Oxygen consumption rate (mg O 2 g DW −1 h −1 ) in juvenile Thenus australiensis.Lobsters were reared at 25.5 ± 0.3 °C and fed 15 N-labeled feed.The ṀO 2 at − 2 h indicates the routine metabolic rate (RMR), and 0 h was set as the first postprandial record.All data represent the mean ± standard error (S.E.) of five individuals.Blue line shows fifth-degree polynomial regression.

Figure 2 .
Figure 2. Total ammonia-N excretion rate (MTAN, mg g DW −1 h −1 ) in juvenile Thenus australiensis.Lobsters were reared at 25.5 ± 0.3 °C and fed 15 N-labeled feed.The MTAN at − 2 h indicates the routine MTAN, and the first postprandial MTAN was recorded at 0 h.All data represent the mean ± standard error (S.E.) of five individuals.Blue line shows sixth-degree polynomial regression.

Figure 3 .
Figure 3. Urea-N excretion rate (Murea-N, µmol g DW −1 h −1 ) in juvenile Thenus australiensis.Lobsters were reared at 25.5 ± 0.3 °C and fed 15 N-labeled feed.The Murea-N at − 2 h indicates the routine Murea-N, and the first postprandial Murea-N was recorded at 0 h.All data represent the mean ± standard error (S.E.) of five individuals.Blue line shows sixth-degree polynomial regression.

Figure 4 .Figure 5 .
Figure 4. Nitrogen quotient (NQ) in juvenile Thenus australiensis.Lobsters were reared at 25.5 ± 0.3 °C and fed 15 N-labeled feed.The NQ at − 2 h indicates the routine NQ, and the first postprandial NQ was recorded at 0 h.The dashed line indicates the theoretical maximum nitrogen quotient of 0.27.All data represent the mean ± standard error (S.E.) of five individuals.

Figure 6 .
Figure 6.Respiratory quotient (RQ) in juvenile Thenus australiensis.Lobsters were reared at 25.5 ± 0.3 °C and fed 15 N-labeled feed.The RQ at − 2 h indicates the routine RQ, and the first postprandial RQ was recorded at 0 h.All data represent the mean ± standard error (S.E.) of five individuals.

Figure 7 .
Figure 7. Instantaneous metabolic energy substrate use (%) in juvenile Thenus australiensis.Lobsters were reared at 25.5 ± 0.3 °C and fed 15 N-labeled feed.The percentage at − 2 h indicates the metabolic energy substrate use during routine metabolism.The first postprandial metabolic energy substrate use was recorded at 0 h.All data represent the mean values of five individuals.

Figure 8 .
Figure 8. Cumulative 15 N-labeled ammonia excretion rate (ce*, %, expressed as the percentage of 15 N in the assimilated feed) in juvenile Thenus australiensis (n = 5) over 48 h post-feeding.Lobsters were reared at 25.5 ± 0.3 °C and fed 15 N-labeled feed.Data points represent the mean ± standard error (S.E.) of five individuals and four sampling times.

Figure 9 .
Figure 9. Fractional whole-body protein synthesis rates (k s % day −1 ) in juvenile Thenus australiensis estimated over 48 h post-feeding.Lobsters were reared at 25.5 ± 0.3 °C and fed 15 N-labeled feed.Data represent the mean ± S.E. of five individuals, p < 0.05.

Figure 10 .
Figure 10.Protein-Nitrogen flux (mg N day −1 ) for a theoretical 50 g WW juvenile Thenus australiensis for the first 24 h following the consumption of a single meal showing a: protein growth (positive protein retention) and b: protein loss (negative protein retention) based on Carter, et al. 44 .