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The current study aimed to investigate the effect of surplus dietary arginine on polyamine and amino acid metabolism and accretion of proteins and lipids, as previous studies have demonstrated a lipid-reducing effect following surplus arginine supplementation in viscera as well as increased muscle growth. Four plant protein-based diets were given increasing concentrations of L-arginine, from 21.1 to 36.1 g kg-1 DM, and fed to quadruplicate tanks of adult Atlantic salmon (1.1 kg) for 12 weeks. No effects on growth or deposition of fat or protein were observed. Free amino acid concentrations and related metabolites were unaffected in the liver, except for urea, while concentrations in muscle and plasma reflected production of arginine metabolites. Polyamine concentrations were unaffected in liver, muscle and white adipose tissue ( WAT), as were the abundance and activity of spermidine/spermine N1-acetyltransferase ( SSAT), the rate-limiting enzyme in polyamine turnover. Gene expression demonstrated differential regulation of ornithine decarboxylase ( ODC) in liver and WAT, although overall little effects were observed on gene expression. Liver S-adenosylmethionine ( SAM) concentrations decreased with arginine supplementation. We suggest that adult Atlantic salmon have adapted to surplus arginine, and the main long-term effect appears to be increased concentrations of arginine metabolites. [ABSTRACT FROM AUTHOR]

Cobia (8.4 ± 0.1 g body weight) were fed to satiation with three test diets of high plant protein-based ingredients and different lysine to arginine ratios, and one commercial diet (currently used for cobia rearing in Vietnam as a control for growth) for 6 weeks. The test diets contained 206 g marine ingredients kg−1, including fishmeal, krill meal and fish protein concentrate (in order of high to low inclusion), while the rest of the dietary protein was a blend of soya and pea protein concentrate, wheat protein and sunflower meal. Crystalline lysine and arginine were added in the test diets to produce either a balanced lysine to arginine ratio ( BL/ A; 1.1) and a high or low lysine to arginine ratio ( HL/ A; 1.8 and LL/ A; 0.8, respectively). There were no significant differences in final body weight, weight gain, feed conversion ratio or protein gain between cobia fed BL/ A- and commercial control diet ( CCT). Cobia fed BL/ A diet performed better than fish fed either HL/ A- or LL/ A diet. This was partly due to a higher feed intake and protein and lipid gain in cobia fed BL/ A diet as compared to HL/ A- and LL/ A diet. [ABSTRACT FROM AUTHOR]

Membrane potential depolarization due to electrogenic peptide transport activity was examined in eel ( Anguilla anguilla) intestinal brush-border membrane vesicles (BBMV) by monitoring the fluorescence quenching of the voltage-sensitive dye 3,3′-diethylthiadicarbocyanine iodide. Our experimental approach consisted of generating an internal negative membrane potential mimicking in vivo conditions and measuring membrane potential depolarization due to different extravesicular dipeptides. Peptide-dependent membrane potential depolarization was observed in both the presence and absence of extravesicular Na+ and was inhibited by diethylpyrocarbonate, which is consistent with the involvement of electrogenic, Na+-independent, H+-dependent peptide transport activity. Kinetic analysis indicated that peptide-dependent membrane potential depolarization is a saturable process ( Km,app ∼ 1.5 mmol L−1) and that within the 0.1–10 mmol L−1 peptide range a single carrier system is involved in the transport process. Our results suggest that a peptide transport activity, kinetically resembling the PepT1(Slc15A1)-type-mediated H+/peptide cotransport action, can be monitored in eel intestinal BBMV using an easy and inexpensive fluorescence assay. [ABSTRACT FROM AUTHOR]

The effect of acute changes of temperature and light on the rates of oxygen consumption and ammonia excretion of developing embryos and yolk-sac larvae of turbot (Scophthalmus maximus ) reared at 15 °C were studied. Short-term temperature adjustments of ±2 °C gave a mean Q [sub 10] of 2.6 for the rate of oxygen consumption. Neither the presence nor the absence of light significantly influenced this response to acute temperature changes, despite light causing a significantly higher rate of oxygen consumption in the yolk-sac larvae between 9 and 12 days after fertilisation. This elevated metabolic rate in the presence of light occurred after pigmentation of the eyes and was probably due to activity associated with food-searching behaviour of this visual feeder. Similarly, for most development, the presence or absence of light and acute temperature adjustments did not cause any significant shift in the relative fraction of amino acids catabolised at 15 °C. However, on day 10 after fertilisation when peak rates of oxygen consumption occurred in the yolk-sac larvae exposed to light, the significantly elevated apparent nitrogen quotients indicated that greater amounts of amino acids were utilised for catabolic substrate oxidation. [ABSTRACT FROM AUTHOR]

Abstract Folate mobilization from the yolk compartment during larval development was studied by analysing the folate concentration in whole body, embryo and yolk in a single batch of Atlantic halibut, Hippoglossus hippoglossus L., eggs and larvae that showed successful fertilization and development. There was a net loss of approx. 50% of folate from yolk during endogenous feeding. Further, only 23% of the decrease in yolk folate was retained in the larval body. The data suggest a need for folate for metabolic and growth purposes during embryogenesis of approximately 2 μ g g-1 weight gain. Relative to these data and published folate requirement for cold-water species, batches of egg from 16 Atlantic halibut brood fish contained variable and, for some batches, critically low levels of folate. This may constitute a potential problem for larval development until start feeding. [ABSTRACT FROM AUTHOR]

At 7 days after first feeding (DAFF), the peptide hormone cholecystokinin (CCK) content (fmol individual−1) and the tryptic activity [μmol arginine-methyl-coumarinyl-7-amide (MCA) min−1 individual−1] per individual gut of Atlantic halibut Hippoglossus hippoglossus larvae were low: 0·2±0·1 and 0·14±0·10, respectively. Thereafter, both parameters increased with the increase in gut mass and reached 19·67±5·58 and 2·71±0·64 at 26 DAFF, respectively. Due to the small sample size, the dry mass (MG, mg) of the individual gut could not be determined accurately at 7 DAFF. At 13 DAFFMG represented 5·5% of whole body dry mass (MW, mg) while at 26 DAFF it had increased to 23%. The mass specific tryptic activity [μmol MCA min−1 per mg dry mass (M)] in the gut increased from 2·74±1·98 at 13 DAFF to 5·00±0·78 at 26 DAFF. There was more individual variation in the mass specific CCK content (fmol M−1) but no significant differences were found, although the data indicated an increase (from 23·38±11·26 at 13 DAFF to 36·27±8·96 fmol M−1 at 26 DAFF). At 7 DAFF the CCK content of the gut represented c. 2% of the whole body CCK content while it increased to c. 62% of the whole body CCK content at 26 DAFF. This demonstrates that it is necessary to separate neural and gastrointestinal sources of CCK in order to determine its alimentary role in fish larvae. Trypsin activity was only found in the gut compartment. In larvae aged 45 DAFF dietary proteins delivery into the gut by tube-feeding appeared to stimulate post-prandial secretion of CCK from the gut as well as stimulate pancreatic trypsin secretion, suggesting that both factors contribute to protein digestion. Copyright 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved. [Copyright &y& Elsevier]

In teleosts, two PepT1-type (Slc15a1) transporters, i.e., PepT1a and PepT1b, are expressed at the intestinal level. They translocate charged di/tripeptides with different efficiency, which depends on the position of the charged amino acid in the peptide and the external pH. The relation between the position of the charged amino acid and the capability of transporting the dipeptide was investigated in the zebrafish and Atlantic salmon PepT1-type transporters. Using selected charged (at physiological pH) dipeptides: i.e., the negatively charged Asp-Gly and Gly-Asp, and the positively charged Lys-Gly and Gly-Lys and Lys-Met and Met-Lys, transport currents and kinetic parameters were collected. The neutral dipeptide Gly-Gln was used as a reference substrate. Atlantic salmon PepT1a and PepT1b transport currents were similar in the presence of Asp-Gly and Gly-Asp, while zebrafish PepT1a elicited currents strongly dependent on the position of Asp in the dipeptide and zebrafish PepT1b elicited small transport currents. For Lys- and Met-containing dipeptides smaller currents compared to Gly-Gln were observed in PepT1a-type transporters. In general, for zebrafish PepT1a the currents elicited by all tested substrates slightly increased with membrane potential and pH. For Atlantic salmon PepT1a, the transport current increased with negative potential but only in the presence of Met-containing dipeptides and in a pH-dependent way. Conversely, large currents were shown for PepT1b for all tested substrates but Gly-Lys in Atlantic salmon. This shows that in Atlantic salmon PepT1b for Lys-containing substrates the position of the charged dipeptides carrying the Lys residue defines the current amplitudes, with larger currents observed for Lys in the N-terminal position. Our results add information on the ability of PepT1 to transport charged amino acids and show species-specificity in the kinetic behavior of PepT1-type proteins. They also suggest the importance of the proximity of the substrate binding site of residues such as Lys PepT1a /Gln PepT1b for recognition and specificity of the charged dipeptide and point out the role of the comparative approach that exploits the natural protein variants to understand the structure and functions of membrane transporters., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vacca, Gomes, De Gennaro, Rønnestad, Bossi and Verri.)

Taurine is a sulphur-containing amino acid with important physiological roles and a key compound for the synthesis of bile salts, which are essential for the emulsion and absorption of dietary lipids. This study aimed to evaluate the effects of taurine supplementation to low-fishmeal diets on the metabolism of taurine, bile acids, and lipids of Senegalese sole. A fishmeal (FM) and a plant-protein-based (PP0) diet were formulated, and the latter was supplemented with taurine at 0.5 and 1.5% (diets PP0.5 and PP1.5). Diets were assigned to triplicate tanks containing 35 fish (initial weight ~14 g) for 6 weeks. Fish from the PP0 treatment presented lower taurine and bile-acid concentrations compared with the FM treatment, and a downregulation of cyp7a1 and abcb11 was observed. Triolein catabolism decreased in PP0-fed fish, resulting in increased hepatic fat content and plasma triglycerides, while no effects on plasma cholesterol were observed. Taurine supplementation to plant-based diets resulted in a higher taurine accumulation in fish tissues, increased bile-acid concentration, and upregulation of cyp7a1 and abcb11 . Hepatic fat content and plasma triglycerides decreased with increasing dietary taurine supplementation. Taurine supplementation mitigated part of the negative effects of plant-based diets, leading to better lipid utilisation.

During the first feeding period, fish will adapt to exogenous feeding as their endogenous source of nutrients is depleted. This requires the development of a functional physiological system to control active search for food, appetite, and food intake. The Atlantic salmon ( Salmo salar ) melanocortin system, a key player in appetite control, includes neuronal circuits expressing neuropeptide y ( npya ), agouti-related peptide ( agrp1 ), cocaine- and amphetamine-regulated transcript ( cart ), and proopiomelanocortin ( pomca ). Little is known about the ontogeny and function of the melanocortin system during early developmental stages. Atlantic salmon [0-730 day degrees (dd)] were reared under three different light conditions (DD, continuous darkness; LD, 14:10 Light: Dark; LL, continuous light) before the light was switched to LD and the fish fed twice a day. We examined the effects of different light conditions (DD LD , LD LD , and LL LD ) on salmon growth, yolk utilization, and periprandial responses of the neuropeptides npya1, npya2, agrp1, cart2a, cart2b, cart4, pomca1 , and pomca2 . Fish were collected 1 week (alevins, 830 dd, still containing yolk sac) and 3 weeks (fry, 991 dd, yolk sac fully consumed) into the first feeding period and sampled before (-1 h) and after (0.5, 1.5, 3, and 6 h) the first meal of the day. Atlantic salmon reared under DD LD , LD LD , and LL LD had similar standard lengths and myotome heights at the onset of first feeding. However, salmon kept under a constant light condition during endogenous feeding (DD LD and LL LD ) had less yolk at first feeding. At 830 dd none of the neuropeptides analyzed displayed a periprandial response. But 2 weeks later, and with no yolk remaining, significant periprandial changes were observed for npya1, pomca1 , and pomca2 , but only in the LD LD fish. This suggests that these key neuropeptides serve an important role in controlling feeding once Atlantic salmon need to rely entirely on active search and ingestion of exogenous food. Moreover, light conditions during early development did not affect the size of salmon at first feeding but did affect the mRNA levels of npya1, pomca1 , and pomca2 in the brain indicating that mimicking natural light conditions (LD LD ) better stimulates appetite control., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Norland, Gomes, Rønnestad, Helvik and Eilertsen.)

In the present study, polyethylene (PE) microplastics (150-300 μm) were added to Atlantic cod (Gadus morhua) feeds at 1 %, either in their present form (Virgin PE) or spiked with PCB-126 (Spiked PE). The feeds were given to juvenile cod for a 4-week period. The fish grew from 11 to 23 g with no significant difference between dietary treatments. Cod fed spiked PE showed a significantly higher concentration of PCB-126 in liver and muscle samples compared to control and fish ingesting virgin PE. In accordance with the accumulation of PCB-126 in the liver, the expression of hepatic cyp1a was higher in cod fed spiked PE. Notably, we observed that spiked PE, as well as virgin PE, have an effect on skin. Overall changes indicated a reduced skin barrier in fish fed a diet containing PE. Indicating that PE itself through interaction with gut tissue may influence skin health in fish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2022 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals LLC.)

Episodes of elevated temperature, combined with lower feed availability, are among the predicted scenarios of climate change representing a challenge for coral reef fish. We investigated the response of clownfish (Amphiprion ocellaris) to a scenario in which it received a single meal to satiety after 48 h fasting at 32 °C (climate change scenario) and 28 °C (control). We analysed the metabolic rate (MR), feed intake, gut transit, and expression of selected brain neuropeptides and one receptor believed to be involved in appetite control. Fish at 32 °C ingested 17.9% less feed and had a faster gut transit than did fish at 28 °C. MR in the unfed fish was 31% higher at 32 °C compared to 28 °C. In the fed fish, postprandial MR at 28 °C was 30% higher compared to that of unfed fish, while at 32 °C it was only 15% higher. The expression of agrp1 did not differ between unfed and refed fish. The levels of both pomca and mc4r increased immediately after the meal and subsequently declined, suggesting a possible anorexic role for these genes. Notably, this pattern was accelerated in fish kept at 32 °C compared with that in fish kept at 28 °C. The dynamics of these changes in expression correspond to a faster gut transition of ingested feed at elevated temperatures. For both agrp2 and pomcb there was an increase in expression following feeding in fish maintained at 32 °C, which was not observed in fish kept at 28 °C. These results suggest that low feed availability and elevated temperature stimulate anorexigenic pathways in clownfish, resulting in significantly lower feed intake despite the temperature-induced increase in metabolic rate. This may be a mechanism to ameliorate the decrease in aerobic scope that results from higher temperatures., Competing Interests: Declaration of Competing Interest Ref.: Ms. No. CBPA-D-22-00190. Metabolic rates, feed intake, appetite control, and gut transit of clownfish Amphiprion ocellaris exposed to increased temperature and limited feed availability. Comparative Biochemistry and Physiology, Part A. All authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

We have described six developmental stages for the ballan wrasse, from the first feeding until the juvenile stage, supported by specific descriptions of cranial ossification, maturation of the digestive tract, and growth-correlated stages. The initial formation and development of bones are closely linked to the functional anatomical structures required for the mechanics of its feeding behavior and ingestion, particularly the jaws and branchial regions involved in opening the mouth and capturing food particles. The overall ontogeny of the cranial structure compares to that of other teleosts. The cranial ossification of the ballan wrasse skull and the development of its dentary apparatus-first pharyngal teeth and later oral teeth-is linked to the development of the digestive system and to their feeding habits, from preying on zooplankton to feeding on crustaceans and invertebrates on rocks and other substrates. As ballan wrasse is a nibbler, eating small meals, the digestive tract is short compared to the length of the fish; there is no stomach or peptic digestion and also no distinctive bulbus and pyloric ceca. The liver and exocrine pancreas and their outlets terminating in the lumen of the most anterior part of the intestine are important in the digestive process and develop with a larger volume than that in gastric teleosts, relative to the digestive system., (© 2022 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

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., (© 2022 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Control of appetite and feed intake in fish larvae are still largely unexplored. Two of the key players in controlling vertebrate's feed intake are cholecystokinin (CCK) and peptide YY (PYY). Here we investigated the mRNA expression of pyy, cck and cck receptors (cckr) in the brain (head) and gut of Atlantic halibut larvae in response to three consecutive meals. We used Artemia nauplii cysts that are commonly ingested by halibut larvae when present as inert feed, and three water-soluble extracts as attractants to stimulate appetite. Cyst intake was not affected by the use of attractants and overall ingestion rate was low. Differences in mRNA expression of cck and pyy were observed between the halibut larvae that had eaten and those that had not despite readily available feed (cysts), supporting that mechanisms for control of feed intake are at least partly functional. All genes analysed were present in the brain and gut, however the different expression profiles between paralogues suggest potential divergent functions. In the gut, cck2 and pyyb mRNA expression was significantly higher in the larvae that ate cysts compared to larvae that decided to not eat, indicating that these genes play a satiety function in the halibut larvae similar to the general vertebrate scheme. However, cck2, cck2r1, and pyy mRNA expression in the brain were lower in the fed-filled larvae group compared to larvae before eating, which contrasts with the presumable anorectic function of these genes. Further research is required to fully evaluate how PYY and CCK affect the feeding biology in halibut larvae, contributing to formulate inert diets that can stimulate appetite and feed intake., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Corticotropin-Releasing Factor (CRF) is one of the main mediators of the Hypothalamic-Pituitary-Interrenal (HPI) axis to stress response. In Atlantic salmon, a comparative understanding of the crf1 paralogs role in the stress response is still incomplete. Our database searches have identified four crf1 genes in Atlantic salmon, named crf1a1, crf1a2, crf1b1 and crf1b2. Brain distribution analysis revealed that the four crf1 paralogs were widely distributed, and particularly abundant in the telencephalon, midbrain, and hypothalamus of Atlantic salmon post-smolts. To increase the knowledge on crf1-mediated response to stress, Atlantic salmon post-smolts were exposed to either repeated chasing, hypoxia or a combination of chasing and hypoxia for eight days, followed by a novel-acute stressor, confinement. Cortisol, glucose, lactate, and creatinine levels were used as markers for the stress response. The crf1 paralogs mRNA abundance showed to be dependent on the stress exposure regime. Both crf1 mRNA levels in the telencephalon and crf1a1 mRNA levels in the hypothalamus showed similar response profiles to the serum cortisol levels, i.e., increasing levels during the first 24 h after stress exposure followed by a decline during the eight-day exposure. The similar trend between crf1 and cortisol disappeared once exposed to the novel-acute stressor. There was a minor response to stress for both crf1b1 and crf1b2 in the hypothalamus, while no changes at mRNA level were observed in the hypothalamic crf1a2 under the different stress conditions. No or weak relationship was found between the crf1 paralogs mRNA expression and the other serum stress-indicators analysed. In summary, our data provide novel insights on the dynamic of the HPI axis activation in Atlantic salmon, and thus underline the involvement of the crf1 paralogs as additional factors in the regulation of the stress response in this species. Likewise, the data highlight the importance of analysing all crf1 paralogues response to a stress-condition, in particular in this premature knowledge stage of their functionality. Further analysis and a more detailed time-point series will help to elucidate the response of the HPI axis and the link of crf1 paralogs in the stress response mechanism., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

System b 0,+ absorbs lysine, arginine, ornithine, and cystine, as well as some (large) neutral amino acids in the mammalian kidney and intestine. It is a heteromeric amino acid transporter made of the heavy subunit SLC3A1/rBAT and the light subunit SLC7A9/b 0,+ AT. Mutations in these two genes can cause cystinuria in mammals. To extend information on this transport system to teleost fish, we focused on the slc3a1 and slc7a9 genes by performing comparative and phylogenetic sequence analysis, investigating gene conservation during evolution (synteny), and defining early expression patterns during zebrafish (Danio rerio) development. Notably, we found that slc3a1 and slc7a9 are non-duplicated in the zebrafish genome. Whole-mount in situ hybridization detected co-localized expression of slc3a1 and slc7a9 in pronephric ducts at 24 h post-fertilization and in the proximal convoluted tubule at 3 days post-fertilization (dpf). Notably, both the genes showed co-localized expression in epithelial cells in the gut primordium at 3 dpf and in the intestine at 5 dpf (onset of exogenous feeding). Taken together, these results highlight the value of slc3a1 and slc7a9 as markers of zebrafish kidney and intestine development and show promise for establishing new zebrafish tools that can aid in the rapid screening(s) of substrates. Importantly, such studies will help clarify the complex interplay between the absorption of dibasic amino acids, cystine, and (large) neutral amino acids and the effect(s) of such nutrients on organismal growth., (© 2021. The Author(s).)

In mammals, knockout of LEPR results in a hyperphagic, morbid obese, and diabetic phenotype, which supports that leptin plays an important role in the control of appetite and energy metabolism, and that its receptor, LEPR, mediates these effects. To date, little is known about the role(s) of lepr in teleost physiology. We investigated a zebrafish (Danio rerio) homozygous lepr knockout (lepr -/- ) line generated by CRISPR/Cas9 in comparison to its wt counterpart with respect to nutrient acquisition, energy allocation, and metabolism. The metabolic characterization included oxygen consumption rate and morphometric parameters (yolk sac area, standard length, wet weight, and condition factor) as proxies for use and allocation of energy in developing (embryos, larvae, and juveniles) zebrafish and showed no particular differences between the two lines, in agreement with previous studies. One exception was found in oxygen consumption at 72 hpf, when zebrafish switch from embryonic to early larval stages and food-seeking behavior could be observed. In this case, the metabolic rate was significantly lower in lepr -/- than in wt. Both phenotypes showed similar responses, with respect to metabolic rate, to acute alterations (22 and 34 °C) in water temperature (measured in terms of Q 10 and activation energy) compared to the standard (28 °C) rearing conditions. To assess lepr involvement in signaling the processing and handling of incoming nutrients when an exogenous meal is digested and absorbed, we conducted an in vivo analysis in lepr -/- and wt early (8 days post-fertilization) zebrafish larvae. The larvae were administered a bolus of protein hydrolysate (0%, 1%, 5%, and 15% lactalbumin) directly into the digestive tract lumen, and changes in the mRNA expression profile before and after (1 and 3 h) administration were quantified. The analysis showed transcriptional differences in the expressions of genes involved in the control of appetite and energy metabolism (cart, npy, agrp, and mc4r), sensing (casr, t1r1, t1r3, t1r2-1, t1r2-2, pept1a, and pept1b), and digestion (cck, pyy, try, ct, and amy), with more pronounced effects observed in the orexigenic than in the anorexigenic pathways, suggesting a role of lepr in their regulations. Differences in the mRNA levels of these genes in lepr -/- vs. wt larvae were also observed. Altogether, our analyses suggest an influence of lepr on physiological processes involved in nutrient acquisition, mainly control of food intake and digestion, during early development, whereas metabolism, energy allocation, and growth seem to be only slightly influenced., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neuropeptide Y (NPY) is known as a potent orexigenic signal in vertebrates, but its role in Atlantic salmon has not yet been fully established. In this study, we identified three npy paralogs, named npya1, npya2, and npyb , in the Atlantic salmon genome. In silico analysis revealed that these genes are well conserved across the vertebrate's lineage and the mature peptide sequences shared at least 77% of identity with the human homolog. We analyzed mRNA expression of npy paralogs in eight brain regions of Atlantic salmon post-smolt, and the effect of 4 days of fasting on the npy expression level. Results show that npya1 was the most abundant paralog, and was predominantly expressed in the telencephalon, followed by the midbrain and olfactory bulb. npya2 mRNA was highly abundant in hypothalamus and midbrain, while npyb was found to be highest expressed in the telencephalon, with low mRNA expression levels detected in all the other brain regions. 4 days of fasting resulted in a significant ( p < 0.05) decrease of npya1 mRNA expression in the olfactory bulb, increased npya2 mRNA expression in the midbrain and decreased npyb mRNA expression in the pituitary. In the hypothalamus, the vertebrate appetite center, expression of the npy paralogs was not significantly affected by feeding status. However, we observed a trend of increased npya2 mRNA expression ( p = 0.099) following 4 days of fasting. Altogether, our findings provide a solid basis for further research on appetite and energy metabolism in Atlantic salmon., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tolås, Kalananthan, Gomes, Lai, Norland, Murashita and Rønnestad.)

Food intake is a vital process that supplies necessary energy and essential nutrients to the body. Information regarding luminal composition in the gastrointestinal tract (GIT) collected through mechanical and nutrient sensing mechanisms are generally conveyed, in both mammals and fish, to the hypothalamic neurocircuits. In this context, ghrelin, the only known hormone with an orexigenic action, and the intestinal peptide transporters 1 and 2, involved in absorption of dietary di- and tripeptides, exert important and also integrated roles for the nutrient uptake. Together, both are potentially involved in signaling pathways that control food intake originating from different segments of the GIT. However, little is known about the role of different paralogs and their response to fasting. Therefore, after 3 weeks of acclimatization, 12 Atlantic salmon ( Salmo salar ) post-smolt were fasted for 4 days to explore the gastrointestinal response in comparison with fed control ( n = 12). The analysis covered morphometric (weight, length, condition factor, and wet content/weight fish %), molecular (gene expression variations), and correlation analyses. Such short-term fasting is a common and recommended practice used prior to any handling in commercial culture of the species. There were no statistical differences in length and weight but a significant lower condition factor in the fasted group. Transcriptional analysis along the gastrointestinal segments revealed a tendency of downregulation for both paralogous genes slc15a1a and slc15a1b and with significant lowered levels in the pyloric ceca for slc15a1a and in the pyloric ceca and midgut for slc15a1b . No differences were found for slc15a2a and slc15a2b (except a higher expression of the fasted group in the anterior midgut), supporting different roles for slc15 paralogs. This represents the first report on the effects of fasting on slc15a2 expressed in GIT in teleosts. Transcriptional analysis of ghrelin splicing variants ( ghrl-1 and ghrl-2 ) showed no difference between treatments. However, correlation analysis showed that the mRNA expression for all genes (restricted to segment with the highest levels) were affected by the residual luminal content. Overall, the results show minimal effects of 4 days of induced fasting in Atlantic salmon, suggesting that more time is needed to initiate a large GIT response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Del Vecchio, Lai, Gomes, Verri, Kalananthan, Barca, Handeland and Rønnestad.)

The clown anemonefish (Amphiprion ocellaris) is a common model species in studies assessing the impact of climate changes on tropical coral fish physiology, metabolism, growth, and stress. However, the basic endocrine principles for the control of food intake and energy homeostasis, under normal and elevated sea temperatures, in this species remain unknown. In this work, we studied food intake and growth in clown anemonefish reared at different temperatures and with different food availability. We also analyzed expression of genes in the melanocortin system, which is believed to be involved in the control of appetite and feeding behavior. These were two paralogues of pomc: pomca and pomcb; two paralogs of agrp: agrp1 and agrp2; and one mc4r-like. Groups of juvenile clown anemonefish were exposed to four experimental treatments combining (orthogonal design) two rearing temperatures: 28 °C (T28; normal) and 32 °C (T32; high) and two feeding regimes: one (1 M; 08:00) or three (3 M; 08:00, 12:00, 15:00) meals per day, fed to satiety by hand. The results showed that high temperature (T32) did not affect the average growth rate but induced a stronger asymmetrical individual body weight of the fish within the population (tank). Lower feeding frequency (1 M) resulted in lower growth rates at both rearing temperatures. Fish reared at high temperature had higher total daily food intake, which correlated with a lower expression of pomca, supporting an anorexigenic role of this gene. High temperature combined with restricted feeding induced higher agrp1 levels and resulted in a higher food intake in the morning meal compared to the control. This supports an orexigenic role for agrp1. mRNA levels of agrp2 responded differently from agrp1, supporting different roles for the paralogues. Levels of mc4r-like inversely correlated with fish body weight, indicating a possible size/stage dependence of gene expression. In conclusion, our results indicate that the melanocortin system is involved in adjusting appetite and food intake of clown anemonefish in response to elevated temperature and low food availability., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The influence of diurnal and nocturnal feeding on daily rhythms of gut levels of cholecystokinin (CCK) and the activity of two key pancreatic proteases, trypsin and chymotrypsin, were examined in juveniles of Senegalese sole (Solea senegalensis), a species with nocturnal habits. Four feeding protocols were performed: P1) One morning meal; P2) Six meals during the light period; P3) Six meals during the dark period; and P4) 12 meals during 24 h. Daily activity patterns of both proteases were remarkably similar and showed a high correlation in all the experimental protocols. In P1, daily patterns of CCK and digestive enzymes showed a single maximum. In P2, CCK levels exhibited two peaks. Digestive enzymes activities showed slightly delayed peaks compared to CCK, although their daily fluctuations were not significant. In P3, intestinal CCK concentration exhibited two peaks at the end of light and dark periods, but only the second one was significant. The first maximum level of chymotrypsin activity occurred 4 h after the first CCK peak, while the second one coincided with the second CCK peak. Fluctuations of trypsin activity were not significant. In P4, CCK concentration showed three small peaks. Digestive enzymes daily fluctuations were not significant, although they showed an inverted trend with respect to CCK. The daily pattern of the gut CCK content in our study is in agreement with the anorexigenic function of this hormone. Our results support the existence of a negative feedback regulatory loop between CCK and pancreatic proteolytic enzymes in Senegalese sole juveniles., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The effects of nutrient and mechanical sensing on gut motility and intestinal metabolism in lower vertebrates remains largely unknown. Here we present the transcriptome response to luminal stimulation by nutrients and an inert bolus on nutrient response pathways and also the response on gut motility in a stomachless fish with a short digestive tract; the ballan wrasse (Labrus berggylta). Using an in vitro model, we differentiate how signals initiated by physical stretch (cellulose and plastic beads) and nutrients (lipid and protein) modulate the gut evacuation rate, motility patterns and the transcriptome. Intestinal stretch generated by inert cellulose initiated a faster evacuation of digesta out of the anterior intestine compared to digestible protein and lipid. Stretch on the intestine upregulated genes associated with increased muscle activity, whereas nutrients stimulated increased expression of several neuropeptides and receptors which are directly involved in gut motility regulation. Although administration of protein and lipid resulted in similar bulbous evacuation times, differences in intestinal motility, transit between the segments and gene expression between the two were observed. Lipid induced increased frequency of ripples and standing contraction in the middle section of the intestine compared to the protein group. We suggest that this difference in motility was modulated by factors [prepronociceptin (pnoca), prodynorphin (pdyn) and neuromedin U (nmu), opioid neurotransmitters and peptides] that are known to inhibit gastrointestinal motility and were upregulated by protein and not lipid. Our findings show that physical pressure in the intestine initiate contractions propelling the bolus distally, directly towards the exit, whereas the stimuli from nutrients modulates the motility to prolong the residence time of digesta in the digestive tract for optimal digestion., Competing Interests: The authors have declared that no competing interests exist.

A tube-feeding model for administering microplastic (MP, Ø = 30 μm) spheres to fish larvae was employed to quantify the uptake of hydrophobic organic contaminants (HOCs) into the larval body through a single administration of MP. Polychlorinated biphenyl-153 (PCB-153) was used as a representative HOC that can be sorbed to MP in the sea. Atlantic herring (Clupea harengus) larvae (34-51 days post-hatching) were selected as the animal model. The herring larvae were tube-fed a single load of up to 200 polystyrene or polyethylene MP spheres spiked with 14 C-labelled PCB-153, and the control larvae were tube-fed an isotonic solution without MP. At the time of sampling (24 h post feeding), some larvae had evacuated all MP spheres from the gut, while others still had MP remaining in the gut. In larvae with a significant number of MP spheres still present in the gut, whole-body scintillation counting (including the MP in the gut lumen) showed elevated levels of the tracer compared to those in the control fish larvae. For larvae in which all or almost all MP had been evacuated by the time of sampling, the tracer levels of the whole body were not significantly different compared to those for the control fish larvae. These data indicate that there was no significant transfer of PCB-153 from contaminated MP into fish larvae within a gut-transit time of <24 h. This study suggests that the vector role of MP in HOC uptake and absorption may be minor compared to that of other HOC uptake pathways., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cationic amino acid transporter 1 (Cat-1 alias Slc7a1) is a Na + -independent carrier system involved in transport and absorption of the cationic amino acids lysine, arginine, histidine, and ornithine and has also been shown to be indispensable in a large variety of biological processes. Starting from isolated full-length zebrafish (Danio rerio) cDNA for slc7a1a, we performed comparative and phylogenetic sequence analysis, investigated the conservation of the gene during vertebrate evolution, and defined tissue expression during zebrafish development. Whole mount in situ hybridization first detected slc7a1a transcripts in somites, eyes, and brain at 14 h post-fertilization (hpf) with additional expression in the distal nephron at 24 hpf and in branchial arches at 3 days post-fertilization (dpf), with significant increase by 5 dpf. Taken together, the expression analysis of the zebrafish Cat-1 system gene slc7a1a suggests a functional role(s) during the early development of the central nervous system, muscle, gills, and kidney. Graphical abstract.

The melanocortin system is a key neuroendocrine network involved in the control of food intake and energy homeostasis in vertebrates. Within the hypothalamus, the system comprises two main distinct neuronal cell populations that express the neuropeptides proopiomelanocortin (POMC; anorexigenic) or agouti-related protein (AGRP; orexigenic). Both bind to the melanocortin-4 receptor (MC4R) in higher order neurons that control both food intake and energy expenditure. This system is relatively well-conserved among vertebrates. However, in Atlantic salmon ( Salmo salar L.), the salmonid-specific fourth round whole-genome duplication led to the presence of several paralog genes which might result in divergent functions of the duplicated genes. In the current study, we report the first comprehensive comparative identification and characterization of Mc4r and extend the knowledge of Pomc and Agrp in appetite control in Atlantic salmon. In silico analysis revealed multiple paralogs for mc4r ( a1 , a2 , b1 , and b2 ) in the Atlantic salmon genome and confirmed the paralogs previously described for pomc ( a1 , a2 , and b ) and agrp ( 1 and 2 ). All Mc4r paralogs are relatively well-conserved with the human homolog, sharing at least 63% amino acid sequence identity. We analyzed the mRNA expression of mc4r , pomc , and agrp genes in eight brain regions of Atlantic salmon post-smolt under two feeding states: normally fed and fasted for 4 days. The mc4ra2 and b1 mRNAs were predominantly and equally abundant in the hypothalamus and telencephalon, the mc4rb2 in the hypothalamus, and a1 in the telencephalon. All pomc genes were highly expressed in the pituitary, followed by the hypothalamus and saccus vasculosus. The agrp genes showed a completely different expression pattern from each other, with prevalent expression of the agrp1 in the hypothalamus and agrp2 in the telencephalon. Fasting did not induce any significant changes in the mRNA level of mc4r , agrp , or pomc paralogs in the hypothalamus or in other highly expressed regions between fed and fasted states. The identification and wide distribution of multiple paralogs of mc4r , pomc , and agrp in Atlantic salmon brain provide new insights and give rise to new questions of the melanocortin system in the appetite regulation in Atlantic salmon., (Copyright © 2020 Kalananthan, Lai, Gomes, Murashita, Handeland and Rønnestad.)