Ana Silva Gomes

Successful transition from endogenous yolk utilization to exogenous feeding is critical for survival in fish larvae, yet the changes in the brain during this transition remain incompletely described. In this study, whole-brain RNA sequencing was used to investigate transcriptomic changes over 48 h during endogenous yolk utilization (720 day degrees (dd)) and after the onset of exogenous feeding (920 dd) in Atlantic salmon ( Salmo salar ), focusing on appetite-related genes. Key components of appetite control, including melanocortin system and nutrient-sensing pathway, were present at 720 dd and elevated levels were observed at 920 dd. Before onset of first feeding, 16 appetite-related genes displayed a significant cyclic profile, where most had a periodicity of 20 h or 28 h. Following the transition from endogenous to exogenous feeding, the majority of significantly cyclic appetite-related genes exhibited a periodicity of 24 h, suggesting the establishment of circadian regulation associated with energy homeostasis. These results indicate that a pre-programmed expression of appetite and energy-related genes occurs in the brain before the yolk is fully utilized. Two weeks into the first feeding period (920 dd), the whole-brain transcriptome showed better responsiveness to feeding, but a fully developed satiety system remained underdeveloped. Additionally, characterization of hypothalamic melanocortin neuropeptides distribution during early salmon development revealed a spatial organization distinct from that reported in later life stages.

Whitefish fisheries’ side‐stream biomass is an abundant underutilized resource that can be valorized to benefit future aquaculture sustainability. Four novel ingredients based on side‐streams from Atlantic cod ( Gadus morhua ) fileting were produced. FM‐hb, a fish meal (FM), and FPH‐hb, a fish protein hydrolysate based on heads (h) and backbones (b); FM‐hbg, a FM based on heads, backbones, and viscera/guts (g); and FPC‐g, a fish protein concentrate based on viscera preserved in formic acid. Four diets were prepared containing one of the ingredients replacing 50% of the dietary FM protein, in addition to a positive (FM10) and a negative (FM5) control. The six diets were fed to triplicate tanks with Atlantic salmon ( Salmo salar L.; 113 ± 1 g) over 8 weeks. Besides general performance, gut and brain gene expression for selected hormones and key neuropeptides involved in the control of appetite and digestive processes were studied during feeding and postprandial, and possible reference levels for Atlantic salmon were established. All side‐stream‐added diets performed well, with no significant differences in performance and biometrics between the treatments. Some gene expression differences were observed, but no well‐defined patterns emerged supporting clear dietary effects related to digestive performance or appetite. However, in the brain, a short‐time upregulation of agouti-related protein-1 ( agrp1 ), corresponded to higher cumulative feed intake (FI) for the FM10 diet supporting notions that this may be a candidate biomarker for appetite in salmon. Expression of stomach ghrelin-1 ( ghrl1 ) was higher than ghrelin-2 ( ghrl2 ) and membrane-bound O-acyltransferase domain-containing 4 ( mboat4 ), and midgut peptide YYa-2 ( pyya2 ) and glucagon-a ( gcga ) were higher than peptide YYb-1 ( pyyb1 ). A comparison showed that midgut peptide YYa-1 ( pyya1 ), pyya2 , and gcga expressions were higher than in the hindgut, which is opposite of what is found in mammals. In conclusion, this study shows that sustainable side‐stream raw materials with different characteristics can partly replace high‐quality commercial FMs giving similar performance.

Zebrafish (Danio rerio) is a well-established animal model, used in a number of research areas. In the last decade, it has also emerged as a tool to evaluate the effects of diets and dietary components and to test novel paradigms in nutrigenomics, nutrigenetics, and nutritional physiology. Despite its worldwide use, the standardization of the zebrafish rearing conditions, including daily nutritional and good feed management practices, is not yet achieved. This is surprising when compared with what is available for other reared animals, such as rodents or other (e.g., commercial) fishes. To date, a major applicative goal in zebrafish nutritional physiology research is to define common, standard, and reproducible protocols of rearing and feeding conditions to generate reliable and comparable results among research laboratories. This review aims to focus on limitations and disadvantages of the current rearing and feeding practices and on some recent technological solutions provided by research groups and/or biotech companies in the field of facility design, with emphasis on automated feeding distribution systems. A general overview of some common schemes of zebrafish husbandry is also given.

The melanocortin system is a key regulator of appetite and food intake in vertebrates. This system includes the neuropeptides neuropeptide y (NPY), agouti‐related peptide (AGRP), cocaine‐ and amphetamine‐regulated transcript (CART), and pro‐opiomelanocortin (POMC). An important center for appetite control in mammals is the hypothalamic arcuate nucleus, with neurons that coexpress either the orexigenic NPY/AGRP or the anorexigenic CART/POMC neuropeptides. In ray‐finned fishes, such a center is less characterized. The Atlantic salmon (Salmo salar) has multiple genes of these neuropeptides due to whole‐genome duplication events. To better understand the potential involvement of the melanocortin system in appetite and food intake control, we have mapped the mRNA expression of npy, agrp, cart, and pomc in the brain of Atlantic salmon parr using in situ hybridization. After identifying hypothalamic mRNA expression, we investigated the possible intracellular coexpression of npy/agrp and cart/pomc in the tuberal hypothalamus by fluorescent in situ hybridization. The results showed that the neuropeptides were widely distributed, especially in sensory and neuroendocrine brain regions. In the hypothalamic lateral tuberal nucleus, the putative homolog to the mammalian arcuate nucleus, npya, agrp1, cart2b, and pomca were predominantly localized in distinct neurons; however, some neurons coexpressed cart2b/pomca. This is the first demonstration of coexpression of cart2b/pomca in the tuberal hypothalamus of a teleost. Collectively, our data suggest that the lateral tuberal nucleus is the center for appetite control in salmon, similar to that of mammals. Extrahypothalamic brain regions might also be involved in regulating food intake, including the olfactory bulb, telencephalon, midbrain, and hindbrain.

The high‐affinity/low‐capacity system Slc15a2 (PepT2) is responsible for the reuptake of di/tripeptides from the renal proximal tubule, but it also operates in many other tissues and organs. Information regarding PepT2 in teleost fish is limited and, to date, functional data are available from the zebrafish (Danio rerio) only. Here, we report the identification of two slc15a2 genes in the Atlantic salmon (Salmo salar) genome, namely slc15a2a and slc15a2b. The two encoded PepT2 proteins share 87% identity and resemble both structurally and functionally the canonical vertebrate PepT2 system. The mRNA tissue distribution analyses reveal a widespread distribution of slc15a2a transcripts, being more abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and the distal part of the gastrointestinal tract. The function of the two transporters was investigated by heterologous expression in Xenopus laevis oocytes and two‐electrode voltage‐clamp recordings of transport and presteady‐state currents. Both PepT2a and PepT2b in the presence of Gly‐Gln elicit pH‐dependent and Na⁺ independent inward currents. The biophysical and kinetic analysis of the recorded currents defined the transport properties, confirming that the two Atlantic salmon PepT2 proteins behave as high‐affinity/low‐capacity transporters. The recent structures and the previous kinetic schemes of rat and human PepT2 qualitatively account for the characteristics of the two Atlantic salmon proteins. This study is the first to report on the functional expression of two PepT2‐type transporters that operate in the same vertebrate organism as a result of (a) gene duplication process(es).Key points: Two slc15a2‐type genes, slc15a2a and slc15a2b coding for PepT2‐type peptide transporters were found in the Atlantic salmon. slc15a2a transcripts, widely distributed in the fish tissues, are abundant in the brain and gills, while slc15a2b transcripts are mainly expressed in the kidney and distal gastrointestinal tract. Amino acids involved in vertebrate Slc15 transport function are conserved in PepT2a and PepT2b proteins. Detailed kinetic analysis indicates that both PepT2a and PepT2b operate as high‐affinity transporters. The kinetic schemes and structures proposed for the mammalian models of PepT2 are suitable to explain the function of the two Atlantic salmon transporters.

The orexigenic agouti-related protein (AgRP) and the anorexigenic pro-opiomelanocortin (POMC) are crucial players in the control of feed intake in vertebrates, yet their role in teleosts has not been fully established. Triplicate groups of Atlantic salmon (Salmo salar) post smolts were subjected to a) fasting for 3 days (fast), and b) normal feeding (fed), resulting in a significant (p < 0.05) upregulation of hypothalamic agrp1 transcripts levels in the fast group. Moreover, the mRNA abundance of agrp1 was significantly (p < 0.05) correlated with the stomach dry weight content. Corresponding inverse patterns were observed for pomca2, albeit not statistically significant. No significant differences were found for the other paralogues, agrp2 and pomca1 and b, between fed and fast groups. The significant correlation between stomach fullness and agrp1 mRNA expression suggests a possible link between the stomach filling/distension and satiety signals. Our study indicates that hypothalamic agrp1 acts as an orexigenic signal in Atlantic salmon.

Peptide transporter 1 (PepT1) mediates the uptake of dietary di-/tripeptides in vertebrates. However, in teleost fish gut, more than one PepT1-type transporter might operate, because of teleost-specific whole gen(om)e duplication event(s) that occurred during evolution. Here, we describe a novel teleost di-/tripeptide transporter, i.e., the Atlantic salmon ( Salmo salar) peptide transporter 1a [PepT1a; or solute carrier family 15 member 1a (Slc15a1a)], which is a paralog (77% similarity and 64% identity at the amino acid level) of the well-described Atlantic salmon peptide transporter 1b [PepT1b, alias PepT1; or solute carrier family 15 member 1b (Slc15a1b)]. Comparative analysis and evolutionary relationships of gene/protein sequences were conducted after ad hoc database mining. Tissue mRNA expression analysis was performed by quantitative real-time PCR, whereas transport function analysis was accomplished by heterologous expression in Xenopus laevis oocytes and two-electrode voltage-clamp measurements. Atlantic salmon pept1a is highly expressed in the proximal intestine (pyloric ceca ≈ anterior midgut > midgut >> posterior midgut), in the same gut regions as pept1b but notably ~5-fold less abundant. Like PepT1b, Atlantic salmon PepT1a is a low‐affinity/high‐capacity system. Functional analysis showed electrogenic, Na⁺-independent/pH-dependent transport and apparent substrate affinity ( K_0.5) values for Gly-Gln of 1.593 mmol/L at pH 7.6 and 0.076 mmol/L at pH 6.5. In summary, we show that a piscine PepT1a-type transporter is functional. Defining the role of Atlantic salmon PepT1a in the gut will help to understand the evolutionary and functional relationships among peptide transporters. Its functional characterization will contribute to elucidate the relevance of peptide transporters in Atlantic salmon nutritional physiology.