Effect of supplementary enzymes on the growth and feed utilisation of gilthead sea bream, Sparus aurata L.

A series of five experiments were carried out to determine the effect of supplementary enzymes on growth performance and feed utilisation of juvenile gilthead sea bream, Sparus aurata, fed diets in which soybean meal (S8M) partially replaced fish meal (FM). In the first of these experiments the addition of cocktails containing 1 g/kg low pH active protease and 1 glkg a-galactosidase or 1 glkg high pH active protease and ] glkg a.-galactosidase to a 320 glkg SBM, 260 glkg FM pressed diet were both found to significantly (P<0.05) improve performance of fish fed these diets compared to fish fed the unsupplemented diet and a 320 glkg FM, 220 glkg SBM diet. This improvement in performance was not obtained when fish were fed 440 glkg SBM, 230 glkg FM diets with the same enzyme combinations. In some parameters performance of fish decreased as the SBM level in the diets was increased. The significant improvements observed in Experiment 1, with addition of enzyme cocktails to the 320 glkg S8M diet, were not repeated in any of the subsequent experiments. The second experiment was aborted due to ahnormal feeding hehaviour of the fish. In the third experiment, in which the enzymes employed in Experiment 1 were used individually at 1 glkg in 320 glkg SBM diets, no significant differences in specific growth rate (SGR), food conversion ratio (FeR) or protein efficiency ratio (PER) were noted in comparison to fish fed the unsupplemented diet. This was also the case with fish fed diets to which the two enzyme cocktails had been added at enzyme inclusion levels of 0.5 glkg each. Although no significant differences were found, feeding the diet with low pH protease alone appeared to increase performance compared to fish fed the unsupplemented diet, and the results of fish fed diets with high pH protease alone or with a.-galactosidase alone indicated that there was a decrease in performance compared to fish fed the unsupplemented diet. In Experiment 4 fish fed 320 g/kg SBM diets with 0.5, 1.0 and 1.5 g/kg low pH protease showed similar SGRs, FCRs and PERs which appeared to show an improved performance (although not significantly so) compared to fish fed diets with 1.0 glkg agalactosidase used together with either 0.5 or 1.0 glkg low pH protease. In the fmal experiment fish were fed 320 g/kg SBM extruded diets to which 0, 0.33, 0.66, 1.00 and 1.33 glkg of low pH protease had been added. Although no significant differences in SGR, FCR or PER were obtained, fish fed the diets containing 0.66 and 1.33 glkg protease appeared to improve performance compared to fish fed any of the other diets or a diet containing 320 glkg FM and 220 glkg SBM. Fish fed the other 320 glkg SBM supplemented dietc; gave similar results. A histological study of the position of nuclei in hepatocytes and the presence of fat globules around hepatopancreatic tissue in liver samples taken from fish fed the various experimental diets failed to show any relationships with either SBM level or enzyme inclusion in the diet. A series of analyses on the distribution of activities of six enzymes in the digestive tract of sea bream indicated that relative activities differed from one enzyme to another and from one region to another. In an investigation into the variation of pH in various parts of the digestive tract after one or two feeds, it was observed that within the first 24 hours after feeding the pH in the stomach decreased to a minimum value of 2.5 and the pH in the rest of the intestine varied between 6.5 and 7.7. vi From a series of gastric evacuation trials which were performed, it was found that the time of day sea bream were fed a meal influenced the gastric evacuation rate, with fish fed in the afternoon taking longer to evacuate the meal than fish fed a similar meal in the morning. Doubling the size of a meal did not double the gastric evacuation time. Instead, the time to evacuate a given percentage of the larger meal only increased by 1.4 and 1.6 times in fish fed the pressed and extruded feeds respectively compared to fish fed the smaller meal. When the sea bream were fed multiple meals it was found that the evacuation rate of an earlier meal was increased by a subsequent meal. A series of trials investigating the distribution in consumption of a population of sea bream fed a single meal indicated that there was a wide variation in the amount of food consumed by each fish in the population and it was observed that even fish of the same size consumed very different quantities of food. Before any definite conclusions can be drawn regarding the use of the three enzymes tested in these experiments to improve growth and feed utilisation in FM-substituted diets, further investigations need to be carried out in an attempt to obtain more significant results. This thesis has shown that additional research into the mode of action of these enzymes is required as well as studies into how the digestive physiology of the sea bream may affect the use of these (and other) supplementary enzymes

Gilthead sea bream

Two investigations were carried out with 150 g gilthead sea bream Sparus aurata to determine the relative activity of six digestive enzymes (pepsin, trypsin, chymotrypsin, carboxypeptidase A, carboxypeptidase B and amylase) and the pH variation in the lumen of different parts of the gut of fish fed one or two meals per day. Pepsin activity was found exclusively in the stomach, whereas activities of the other enzymes studied were found in all regions of the gut, including the stomach. The lack of localization of enzyme production in the digestive tract of S. aurata is similar to many other species as reported in the literature. The pH variations found in the different regions of the gut could be explained by general digestive physiology following the flow of digesta along the digestive tract. The range of pHs recorded in the various regions of the gut were generally outside the cited optima for many digestive proteases in this species.peer-reviewe

A multitrophic culture system for the production of black soldier fly larvae (Hermetia illucens)

Goals number 2, 11 and 12 of the 17 sustainable development goals, enacted by the United Nations as part of the 2030 Agenda for sustainable development, aim to end hunger as a priority, create sustainable cities and above all encourage responsible consumption and production. With increasing world population and higher demand for food, we need to find ways of producing cheap sources of protein and lipid that may in turn be used as animal or aquaculture feed. A multitrophic system involving mealworm larvae (MWL, Tenebrio molitor) and black soldier fly larvae (BSFL, Hermetia illucens) was developed to transform fruit and vegetable kitchen waste into usable biomass. MWL, fed mainly on kitchen waste, reached an average prepupal length of 2.4 cm, fresh weight of 0.12 g and dry matter protein and lipid contents of 44.2% and 16.5% respectively, with an average specific growth rate (SGR) of 2.2%/day and a feed conversion ratio (FCR) of 7.9. Conversely, BSFL fed on a variety of feeds, including MWL frass, kitchen waste and oats, had an average prepupal length of 1.3 cm, fresh weight of 0.16 g and dry matter protein and lipid contents of 41.4% and 26.3% respectively, with an average SGR and FCR of 4.3%/day and 8.9 respectively. The BSFL fed MWL frass obtained some of highest SGR and lowest FCR values, with one group achieving 7.5%/day and 2.9 respectively. This investigation has demonstrated the feasibility of a multi-trophic production system using kitchen waste to feed MWL whose frass was in turn used to feed BSFL thereby producing protein- and lipid-rich biomass that can serve as animal or aquaculture feed.peer-reviewe

A multitrophic culture system for the production of black soldier fly larvae (Hermetia illucens)

A multitrophic system involving mealworm larvae (MWL) and black soldier fly larvae (BSFL) was developed to transform kitchen waste into usable biomass. MWL, fed mainly on kitchen waste, reached an average prepupal length of 2.4cm, fresh weight of 0.12g and protein and lipid content (dry matter) of 44.2% and 16.5% respectively, with an average specific growth rate (SGR) of 2.2%/day and a feed conversion ratio (FCR) of 7.9. The frass generated by the MWL was collected and subsequently fed to the BSFL for an average period of 21 days. On average each MWL, weighing about 0.13g, produced 0.14g of frass over an average period of 97 days. Different batches of BSFL, fed on a variety of feeds, including the MWL frass, kitchen waste and oats, had an average prepupal length of 1.3cm, fresh weight of 0.16g and protein and lipid contents (dry matter) of 41.4% and 26.3% respectively, with an average SGR and FCR of 4.3%/day and 8.9 respectively. BSFL fed MWL frass only obtained some of highest SGR values and the best FCR, with one group achieving 7.5%/day and 2.9 respectively. The amount of frass generated by the MWL, a total of 646g, was capable of sustaining 253.0g of prepupal stage BSFL. This preliminary investigation has demonstrated that a multi-trophic production system using kitchen waste-fed MWL frass to feed BSFL is feasible. Further studies should be carried out to see if the multitrophic concept can be exploited as a production approach.peer-reviewe

Use of waste polystyrene as feed for mealworms (Tenebrio molitor)

This study aimed to determine what percentage of the feed provided to the mealworm larvae of Tenebrio molitor (MWL) could be substituted by polystyrene (PS) whilst retaining mealworm viability. The survival and growth rates, as well as the general performance of the larvae, were followed for a 6-week duration. Four test groups in three independent replicates of MWL, each with 300 larvae at the 8th instar, were fed on varying PS-bran percentages for six weeks under standard growth environment conditions. The four feeding regimes were 0, 50, 75 and 100% PS. The remainder of the feed for the 0, 50 and 75% groups was wheat bran. A 5g carrot supplement was provided for all groups weekly. The mealworms were monitored for mortality, percentage weight gain (%WG) and percentage length gain (%LG), and changes in their lipid and protein content at the end of the 6-week trial. Feeding solely PS did not provide the best degradation of PS, with only 27.39% degradation in six weeks. The 75% PS treatment had the lowest %PS consumption at 21.74%. The best alternative was to provide a feed consisting of 50-50 PS-wheat bran supplemented with carrots giving the highest %PS consumption at 31.06%. Furthermore, over six weeks, the 50% PS treatment larvae had the best %LG and %WG at 47.1% and 278.6%, respectively. By comparison, the 100% PS treatment achieved a %LG and %WG of 36.6% and 205.4 respectively. Mortalities of the 50% PS treatment were of 54.7%, similar to the 75% PS group, whilst, for the 100% PS, mortalities were 73.89%. The control group achieved the lowest mortalities at 22.78%. An additional test was conducted to investigate if a diet consisting of 50% PS significantly affected the protein and lipid content of the MWL compared to the control group. Analysis showed a statistically significant difference in protein content, with the 50% PS diet resulting in a higher protein content of 56.49% and 25.51 % lipid content, on a dry matter basis, when compared to the control group. In conclusion, the 50% PS treatment larvae gave the best larval growth and survival rates out of the three treatments with PS provided in their diet, whilst the 100% PS gave poor results as PS degradation was not as efficient in this test group. A better approach to making use of waste PS is actually to provide the PS as part of a diet supplemented with natural feeds, such as wheat bran and carrots. For the commercialising of this concept, further studies need to be conducted.peer-reviewe

Thermal sensitivity of field metabolic rate predicts differential futures for bluefin tuna juveniles across the Atlantic Ocean

Changing environmental temperatures impact the physiological performance of fishes, and consequently their distributions. A mechanistic understanding of the linkages between experienced temperature and the physiological response expressed within complex natural environments is often lacking, hampering efforts to project impacts especially when future conditions exceed previous experience. In this study, we use natural chemical tracers to determine the individual experienced temperatures and expressed field metabolic rates of Atlantic bluefin tuna (Thunnus thynnus) during their first year of life. Our findings reveal that the tuna exhibit a preference for temperatures 2–4 °C lower than those that maximise field metabolic rates, thereby avoiding temperatures warm enough to limit metabolic performance. Based on current IPCC projections, our results indicate that historically-important spawning and nursery grounds for bluefin tuna will become thermally limiting due to warming within the next 50 years. However, limiting global warming to below 2 °C would preserve habitat conditions in the Mediterranean Sea for this species. Our approach, which is based on field observations, provides predictions of animal performance and behaviour that are not constrained by laboratory conditions, and can be extended to any marine teleost species for which otoliths are available

Thermal sensitivity of field metabolic rate predicts differential futures for bluefin tuna juveniles across the Atlantic Ocean

Changing environmental temperatures impact the physiological performance of fishes, and consequently their distributions. A mechanistic understanding of the linkages between experienced temperature and the physiological response expressed within complex natural environments is often lacking, hampering efforts to project impacts especially when future conditions exceed previous experience. In this study, we use natural chemical tracers to determine the individual experienced temperatures and expressed field metabolic rates of Atlantic bluefin tuna (Thunnus thynnus) during their first year of life. Our findings reveal that the tuna exhibit a preference for temperatures 2–4 °C lower than those that maximise field metabolic rates, thereby avoiding temperatures warm enough to limit metabolic performance. Based on current IPCC projections, our results indicate that historically-important spawning and nursery grounds for bluefin tuna will become thermally limiting due to warming within the next 50 years. However, limiting global warming to below 2 °C would preserve habitat conditions in the Mediterranean Sea for this species. Our approach, which is based on field observations, provides predictions of animal performance and behaviour that are not constrained by laboratory conditions, and can be extended to any marine teleost species for which otoliths are available

Spatial dynamics and mixing of bluefin tuna in the Atlantic Ocean and Mediterranean Sea revealed using next generation sequencing

The Atlantic bluefin tuna is a highly migratory species emblematic of the challenges associated with shared fisheries management. In an effort to resolve the species’ stock dynamics, a genomewide search for spatially informative single nucleotide polymorphisms (SNPs) was undertaken, by way of sequencing reduced representation libraries. An allele frequency approach to SNP discovery was used, combining the data of 555 larvae and young-of-the-year (LYOY) into pools representing major geographical areas and mapping against a newly assembled genomic reference. From a set of 184,895 candidate loci, 384 were selected for validation using 167 LYOY. A highly discriminatory genotyping panel of 95 SNPs was ultimately developed by selecting loci with the most pronounced differences between western Atlantic and Mediterranean Sea LYOY. The panel was evaluated by genotyping a different set of LYOY (n = 326), and from these, 77.8% and 82.1% were correctly assigned to western Atlantic and Mediterranean Sea origins, respectively. The panel revealed temporally persistent differentiation among LYOY from the western Atlantic and Mediterranean Sea (FST = 0.008, p = .034). The composition of six mixed feeding aggregations in the Atlantic Ocean and Mediterranean Sea was characterized using genotypes from medium (n = 184) and large (n = 48) adults, applying population assignment and mixture analyses. The results provide evidence of persistent population structuring across broad geographic areas and extensive mixing in the Atlantic Ocean, particularly in the mid-Atlantic Bight and Gulf of St. Lawrence. The genomic reference and genotyping tools presented here constitute novel resources useful for future research and conservation efforts

Validation of the Growth Equation Applicable to the Eastern Atlantic Bluefin Tuna, Thunnus thynnus (L.), Using Lmax, Tag-Recapture, and First Dorsal Spine Analysis

The growth equation currently used for Atlantic bluefin tuna, Thunnus thynnus (L.), eastern stock (Lt = 318.85 [1-e^-0.093(t + 0.97)]) is validated using several approaches. The first method involved a comparison of studies with von Bertalanffy parameter estimates in which, different methods for the age estimation are utilized, taking as references the maximum size of this species (Lmax = 319.93 +- 11.3 cm) and the growth equation of the western Atlantic stock (Lt = 314.90 [1-e^-0.089 (t+ 1.13)]). The result of this analysis showed that the growth equation used by ICCAT s Standing Committee on Research and Statistics Atlantic bluefin tuna assessment group for the eastern stock perfectly fits Lmax. Second, an analysis was realized from first dorsal spine rings, 578 samples (age groups 0 to 3) of ABFT collected from the Bay of Biscay and the Mediterranean Sea, enabled the interpretation of the wide opaque bands (fast growth), formed during the boreal late spring and completed by autumn (June to November), and the translucent rings (hyaline rings, slow growth), formed during boreal autumn to late spring (November to May-June). In addition, first dorsal spine sections bands of two recovered fish that had carried conventional and electronic archival tags are also consistent. The chronological analysis of the opaque bands and hyaline rings of one fish tagged with an archival tag and recovered in the Bay of Biscay (the first time such a spine had been available for such analysis) revealed that transatlantic migrations may lead to double hyaline ring formation in the spine. Finally, the validation of the ABFT growth equation is made by superimposing tag-recovery data from tagging surveys in the Bay of Biscay, western Mediterranean and western Atlantic (N = 131) and spine readings (N = 299) to the eastern stock ABFT growth equation and analysing residuals. The coefficient of determination (R2 = 97.98) and the residual s distribution indicated good performance of the model. Although no important differences between the growth model of the eastern stock and that of the western stock are found, in all cases studied, the predictive accuracy indicators are better for the eastern model.The authors thank the European Data Collection Program (ROJOCAN2007) and the Basque Government for the financing of the recent tagging campaigns.Cort, JL.; Arregui, I.; Estruch, V.; Deguara, S. (2014). Validation of the Growth Equation Applicable to the Eastern Atlantic Bluefin Tuna, Thunnus thynnus (L.), Using Lmax, Tag-Recapture, and First Dorsal Spine Analysis. 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Life History and Stock Structure of Atlantic Bluefin Tuna (Thunnus thynnus). Reviews in Fisheries Science, 15(4), 265-310. doi:10.1080/10641260701484135Santamaria, N., Bello, G., Corriero, A., Deflorio, M., Vassallo-Agius, R., Bök, T., & De Metrio, G. (2009). Age and growth of Atlantic bluefin tuna,Thunnus thynnus(Osteichthyes: Thunnidae), in the Mediterranean Sea. Journal of Applied Ichthyology, 25(1), 38-45. doi:10.1111/j.1439-0426.2009.01191.xWestman, J. R., & Gilbert, P. W. (1941). Notes on Age Determination and Growth of the Atlantic Bluefin Tuna, Thunnus thynnus (Linnaeus). Copeia, 1941(2), 70. doi:10.2307/143743