Final Report Summary - GASTRONEUROPEP (Gastrointestinal and neuropeptides regulating food intake in fish in response to dietary lipid composition)
The aquaculture industry is facing one of its biggest challenges, due to the necessity to replace fish meal (FM) and fish oil (FO) in fish diets to meet escalating demands for aquaculture feeds, associated with rises in world aquaculture production, while fisheries landings (traditionally supplying raw materials for aquafeeds) are stable or declining. Therefore, fish diet formulations have changed dramatically in recent years, particularly with the inclusion of increasing amounts of vegetable ingredients (vegetable meal - VM - and vegetable oils - VO). Intense research has been directed towards understanding and mitigating potential negative effects of these dietary changes, in particular related to impacts on fish health, metabolism and product quality. However, even though there is some evidence suggesting that vegetable-based diets might have poorer acceptability and reduce food intake, little has been investigated in detail in this area. Furthermore, very little information exists on the mechanisms regulating food intake in fish, which is paradoxical considering how this is probably one of the most important factors affecting fish growth and that feed is the single highest running cost in any aquaculture operation.
Having this in mind, the objective of this project was to evaluate potential impacts of alternative feed formulations containing VO on food intake and, by investigating in parallel changes in the expression of key gastrointestinal and neuropeptides that are likely implicated in the regulation of appetite, attempt to identify possible regulatory mechanisms which might be affected by these dietary replacements changing the diet's fatty acid composition. In addition, effects on several lipid metabolism genes were also analysed to study possible interrelations between energy homeostasis, lipid sensing and appetite in fish. Studies were performed with two species of high aquaculture interest, the rainbow trout and Senegalese sole and, additionally, for Senegalese sole the effects of fatty acid composition on food intake and lipid metabolism were investigated at different life stages (larvae and juveniles) that have very distinct requirements. Some studies employed a typical nutritional approach (testing different diet formulations) but others employed a more physiological methodology (tube feeding or intraperitoneal injections of pure fatty acids), in both cases combined with molecular analyses (gene expression).
Although work is still ongoing, data collected so far indicates that important progress has been made, especially since little information existed in this field. We now have some clues on how the lipid composition of the diet might affect different metabolic pathways and particularly which neuropeptides appear to respond transcriptionally to the tested dietary changes. In general, it appears that the dietary replacement of FO by VO did not substantially affect food intake in juveniles of a freshwater (trout) and marine (sole) fish species, and in trout the replacement of FM by VM had a more significant effect. However, it is likely that other marine fish species have higher sensitivity to replacement of FO and FM by vegetable alternatives than Senegalese sole since it recently became clear that sole are unique among marine aquaculture species as they can biosynthesize DHA and therefore can likely tolerate higher levels of vegetable ingredients in their diet (work conducted in parallel by the fellow during the period of the ERG). In fish larvae there are very few studies on food intake, given the technical difficulty in accurately measuring ingestion of live preys during the larval stages. In this project we specifically developed a technique to measure food intake using fluorescent microspheres to label the diet and have unequivocally established that there can be differences in ingestion of live preys depending on their lipid enrichment which are not associated with prey selectivity, i.e. independent of visual or sensorial cues. In addition, important information was collected for both sole larvae and juveniles regarding the timing of response of the target genes, i.e. whether differences can be observed in basal conditions (in fasted animals) and how their transcription rates respond to feeding, at different times after feeding. In general, a fast and short-term effect was measured, as most changes in gene expression occurred only 30 mins after feeding in larvae and from 1 to 3 h in sole juveniles. Finally, we obtained new information on sole neuropeptides, and in some cases uncovered and characterized different related genes with respect to their tissue distribution, mapping of their expression in different regions of the brain, and transcriptional response to feeding and dietary composition. One of the most intriguing aspects of the project was that the observed changes in the levels of expression of some of the neuropeptides were not always what would be expected having in mind their putative anorexigenic (i.e. depressing appetite) or orexigenic (i.e. stimulating appetite) role and the observed effects of diets in terms of feed intake. This indicates that further information is necessary on the mode of operation of these genes and on these complex regulatory networks in order to understand how changes in gene expression can ultimately affect appetite and feeding in fish. In this respect, the project has originated new information on qPCR primers and assay conditions for several important appetite- and metabolism-related Senegalese sole genes, which should stimulate future research in this field.
In conclusion, a main outcome of this project was to contribute to establishing essential research tools and collected basic information which will enable to zoom in on questions of high scientific and practical (for the development of aquaculture diets including alternative ingredients) interest in the future. We believe that this will have considerable impact in shaping future research in this emergent field.
Having this in mind, the objective of this project was to evaluate potential impacts of alternative feed formulations containing VO on food intake and, by investigating in parallel changes in the expression of key gastrointestinal and neuropeptides that are likely implicated in the regulation of appetite, attempt to identify possible regulatory mechanisms which might be affected by these dietary replacements changing the diet's fatty acid composition. In addition, effects on several lipid metabolism genes were also analysed to study possible interrelations between energy homeostasis, lipid sensing and appetite in fish. Studies were performed with two species of high aquaculture interest, the rainbow trout and Senegalese sole and, additionally, for Senegalese sole the effects of fatty acid composition on food intake and lipid metabolism were investigated at different life stages (larvae and juveniles) that have very distinct requirements. Some studies employed a typical nutritional approach (testing different diet formulations) but others employed a more physiological methodology (tube feeding or intraperitoneal injections of pure fatty acids), in both cases combined with molecular analyses (gene expression).
Although work is still ongoing, data collected so far indicates that important progress has been made, especially since little information existed in this field. We now have some clues on how the lipid composition of the diet might affect different metabolic pathways and particularly which neuropeptides appear to respond transcriptionally to the tested dietary changes. In general, it appears that the dietary replacement of FO by VO did not substantially affect food intake in juveniles of a freshwater (trout) and marine (sole) fish species, and in trout the replacement of FM by VM had a more significant effect. However, it is likely that other marine fish species have higher sensitivity to replacement of FO and FM by vegetable alternatives than Senegalese sole since it recently became clear that sole are unique among marine aquaculture species as they can biosynthesize DHA and therefore can likely tolerate higher levels of vegetable ingredients in their diet (work conducted in parallel by the fellow during the period of the ERG). In fish larvae there are very few studies on food intake, given the technical difficulty in accurately measuring ingestion of live preys during the larval stages. In this project we specifically developed a technique to measure food intake using fluorescent microspheres to label the diet and have unequivocally established that there can be differences in ingestion of live preys depending on their lipid enrichment which are not associated with prey selectivity, i.e. independent of visual or sensorial cues. In addition, important information was collected for both sole larvae and juveniles regarding the timing of response of the target genes, i.e. whether differences can be observed in basal conditions (in fasted animals) and how their transcription rates respond to feeding, at different times after feeding. In general, a fast and short-term effect was measured, as most changes in gene expression occurred only 30 mins after feeding in larvae and from 1 to 3 h in sole juveniles. Finally, we obtained new information on sole neuropeptides, and in some cases uncovered and characterized different related genes with respect to their tissue distribution, mapping of their expression in different regions of the brain, and transcriptional response to feeding and dietary composition. One of the most intriguing aspects of the project was that the observed changes in the levels of expression of some of the neuropeptides were not always what would be expected having in mind their putative anorexigenic (i.e. depressing appetite) or orexigenic (i.e. stimulating appetite) role and the observed effects of diets in terms of feed intake. This indicates that further information is necessary on the mode of operation of these genes and on these complex regulatory networks in order to understand how changes in gene expression can ultimately affect appetite and feeding in fish. In this respect, the project has originated new information on qPCR primers and assay conditions for several important appetite- and metabolism-related Senegalese sole genes, which should stimulate future research in this field.
In conclusion, a main outcome of this project was to contribute to establishing essential research tools and collected basic information which will enable to zoom in on questions of high scientific and practical (for the development of aquaculture diets including alternative ingredients) interest in the future. We believe that this will have considerable impact in shaping future research in this emergent field.