Functional characterization of Atlantic salmon (Salmo salar L.) PepT2 transporters.

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.

Developmental stages of the ballan wrasse from first feeding through metamorphosis: Cranial ossification and the digestive system.

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.

Metabolic rates, feed intake, appetite control, and gut transit of clownfish Amphiprion ocellaris exposed to increased temperature and limited feed availability.

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.

Food intake, growth, and expression of neuropeptides regulating appetite in clown anemonefish (Amphiprion ocellaris) exposed to predicted climate changes.

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.

Leptin receptor-deficient (knockout) zebrafish: Effects on nutrient acquisition.

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 Q10 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.

The stress response in Atlantic salmon (Salmo salar L.): identification and functional characterization of the corticotropin-releasing factor (crf) paralogs.

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.

Daily rhythms of intestinal cholecystokinin and pancreatic proteases activity in Senegalese sole juveniles with diurnal and nocturnal feeding.

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.

The zebrafish cationic amino acid transporter/glycoprotein-associated family: sequence and spatiotemporal distribution during development of the transport system b0,+ (slc3a1/slc7a9).

System b0,+ 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/b0,+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.

Identification and characterization of the Atlantic salmon peptide transporter 1a.

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 (K0.5) 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.

Sequence analysis and spatiotemporal developmental distribution of the Cat-1-type transporter slc7a1a in zebrafish (Danio rerio).

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.

Loss of stomach, loss of appetite? Sequencing of the ballan wrasse (Labrus bergylta) genome and intestinal transcriptomic profiling illuminate the evolution of loss of stomach function in fish.

BACKGROUND: The ballan wrasse (Labrus bergylta) belongs to a large teleost family containing more than 600 species showing several unique evolutionary traits such as lack of stomach and hermaphroditism. Agastric fish are found throughout the teleost phylogeny, in quite diverse and unrelated lineages, indicating stomach loss has occurred independently multiple times in the course of evolution. By assembling the ballan wrasse genome and transcriptome we aimed to determine the genetic basis for its digestive system function and appetite regulation. Among other, this knowledge will aid the formulation of aquaculture diets that meet the nutritional needs of agastric species. RESULTS: Long and short read sequencing technologies were combined to generate a ballan wrasse genome of 805 Mbp. Analysis of the genome and transcriptome assemblies confirmed the absence of genes that code for proteins involved in gastric function. The gene coding for the appetite stimulating protein ghrelin was also absent in wrasse. Gene synteny mapping identified several appetite-controlling genes and their paralogs previously undescribed in fish. Transcriptome profiling along the length of the intestine found a declining expression gradient from the anterior to the posterior, and a distinct expression profile in the hind gut. CONCLUSIONS: We showed gene loss has occurred for all known genes related to stomach function in the ballan wrasse, while the remaining functions of the digestive tract appear intact. The results also show appetite control in ballan wrasse has undergone substantial changes. The loss of ghrelin suggests that other genes, such as motilin, may play a ghrelin like role. The wrasse genome offers novel insight in to the evolutionary traits of this large family. As the stomach plays a major role in protein digestion, the lack of genes related to stomach digestion in wrasse suggests it requires formulated diets with higher levels of readily digestible protein than those for gastric species.

The two-step development of a duplex retina involves distinct events of cone and rod neurogenesis and differentiation.

Unlike in mammals, persistent postembryonic retinal growth is a characteristic feature of fish, which includes major remodeling events that affect all cell types including photoreceptors. Consequently, visual capabilities change during development, where retinal sensitivity to different wavelengths of light (photopic vision), -and to limited photons (scotopic vision) are central capabilities for survival. Differently from well-established model fish, Atlantic cod has a prolonged larval stage where only cone photoreceptors are present. Rods do not appear until juvenile transition (metamorphosis), a hallmark of indirect developing species. Previously we showed that whole gene families of lws (red-sensitive) and sws1 (UV-sensitive) opsins have been lost in cod, while rh2a (green-sensitive) and sws2 (blue-sensitive) genes have tandem duplicated. Here, we provide a comprehensive characterization of a two-step developing duplex retina in Atlantic cod. The study focuses on cone subtype dynamics and delayed rod neurogenesis and differentiation in all cod life stages. Using transcriptomic and histological approaches we show that different opsins disappear in a topographic manner during development where central to peripheral retina is a key axis of expressional change. Early cone differentiation was initiated in dorso-temporal retina different from previously described in fish. Rods first appeared during initiation of metamorphosis and expression of the nuclear receptor transcription factor nr2e3-1, suggest involvement in rod specification. The indirect developmental strategy thus allows for separate studies of cones and rods development, which in nature correlates with visual changes linked to habitat shifts. The clustering of key retinal genes according to life stage, suggests that Atlantic cod with its sequenced genome may be an important resource for identification of underlying factors required for development and function of photopic and scotopic vision.

Involvement of cholecystokinin (CCK) in the daily pattern of gastrointestinal regulation of Senegalese sole (Solea senegalensis) larvae reared under different feeding regimes.

Cholecystokinin (CCK) is an important regulator of pancreatic enzyme secretion in adult mammals and teleosteans. Although some studies have focused on the interaction between CCK and trypsin in marine fish larvae, little is known about the circadian patterns of the regulatory mechanism involving these two digestive components. In this study, we took advantage of the characteristic change from a diurnal to a nocturnal feeding habit that occurs in Senegalese sole (Solea senegalensis) post-larvae, to conduct an experiment where larvae and postlarvae were submitted to three different feeding regimes from mouth opening: continuous feeding, diurnal feeding and nocturnal feeding. The aim was to establish different daily feeding scenarios to uncover the operating mechanisms of CCK and tryptic enzyme activity over the 24-hourcycle to better understand the regulation of digestion in developing fish larvae. Results show a prevalence of simultaneous and opposing trends of CCK level and tryptic activity as a function of the postprandial time. This finding supports the existence of a regulatory loop between these two digestive components in pre- and post-metamorphic Senegal sole larvae. In addition, CCK level was also modulated by the gut content, tending to be lower when the gut is full and higher when is being emptied. Furthermore, larvae were able to synchronize digestive functions to very different feeding regimes, although it seems to be important having a diurnal feeding phase during pre-metamorphic stages for a proper development.

The transcriptome of metamorphosing flatfish.

BACKGROUND: Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. Metamorphosis in vertebrates is driven by thyroid hormones (THs), but how they orchestrate the cellular, morphological and functional modifications associated with maturation to juvenile/adult states in flatfish is an enigma. Since THs act via thyroid receptors that are ligand activated transcription factors, we hypothesized that the maturation of tissues during metamorphosis should be preceded by significant modifications in the transcriptome. Targeting the unique metamorphosis of flatfish and taking advantage of the large size of Atlantic halibut (Hippoglossus hippoglossus) larvae, we determined the molecular basis of TH action using RNA sequencing. RESULTS: De novo assembly of sequences for larval head, skin and gastrointestinal tract (GI-tract) yielded 90,676, 65,530 and 38,426 contigs, respectively. More than 57 % of the assembled sequences were successfully annotated using a multi-step Blast approach. A unique set of biological processes and candidate genes were identified specifically associated with changes in morphology and function of the head, skin and GI-tract. Transcriptome dynamics during metamorphosis were mapped with SOLiD sequencing of whole larvae and revealed greater than 8,000 differentially expressed (DE) genes significantly (p < 0.05) up- or down-regulated in comparison with the juvenile stage. Candidate transcripts quantified by SOLiD and qPCR analysis were significantly (r = 0.843; p < 0.05) correlated. The majority (98 %) of DE genes during metamorphosis were not TH-responsive. TH-responsive transcripts clustered into 6 groups based on their expression pattern during metamorphosis and the majority of the 145 DE TH-responsive genes were down-regulated. CONCLUSIONS: A transcriptome resource has been generated for metamorphosing Atlantic halibut and over 8,000 DE transcripts per stage were identified. Unique sets of biological processes and candidate genes were associated with changes in the head, skin and GI-tract during metamorphosis. A small proportion of DE transcripts were TH-responsive, suggesting that they trigger gene networks, signalling cascades and transcription factors, leading to the overt changes in tissue occurring during metamorphosis.

First feed affects the expressions of microRNA and their targets in Atlantic cod.

To our knowledge, there is no report on microRNA (miRNA) expression and their target analysis in relation to the type of the first feed and its effect on the further growth of fish. Atlantic cod (Gadus morhua) larvae have better growth and development performance when fed natural zooplankton as a start-feed, as compared with those fed typical aquaculture start-feeds. In our experiment, two groups of Atlantic cod larvae were fed reference feed (zooplankton, mostly copepods, filtered from a seawater pond) v. aquaculture feeds: enriched rotifers (Brachionus sp.) and later brine shrimp (Artemia salina). We examined the miRNA expressions of six defined developmental stages as determined and standardised by body length from first feeding for both diet groups. We found eight miRNA (miR-9, miR-19a, miR-130b, miR-146, miR-181a, miR-192, miR-206 and miR-11240) differentially expressed between the two feeding groups in at least one developmental stage. We verified the next-generation sequencing data using real-time RT-PCR. We found 397 putative targets (mRNA) to the differentially expressed miRNA; eighteen of these mRNA showed differential expression in at least one stage. The patterns of differentially expressed miRNA and their putative target mRNA were mostly inverse, but sometimes also concurrent. The predicted miRNA targets were involved in different pathways, including metabolic, phototransduction and signalling pathways. The results of this study provide new nutrigenomic information on the potential role of miRNA in mediating nutritional effects on growth during the start-feeding period in fish larvae.

Identification of a novel leptin receptor duplicate in Atlantic salmon: Expression analyses in different life stages and in response to feeding status.

In recent years rapidly growing research has led to identification of several fish leptin orthologs and numerous duplicated paralogs possibly arisen from the third and fourth round whole genome duplication (3R and 4R WGD) events. In this study we identify in Atlantic salmon a duplicated LepRA gene, named LepRA2, that further extend possible evolutionary scenarios of the leptin and leptin receptor system. The 1121 amino acid sequence of the novel LepRA2 shares 80% sequence identity with the LepRA1 paralog, and contains the protein motifs typical of the functional (long form) leptin receptor in vertebrates. In silico predictions showed similar electrostatic properties of LepRA1 and LepRA2 and high sequence conservation at the leptin interaction surfaces within the CHR/leptin-binding and FNIII domains, suggesting conserved functional specificity between the two duplicates. Analysis of temporal expression profiles during pre-hatching stages indicate that both transcripts are involved in modulating leptin developmental functions, although the LepRA1 paralog may play a major role as the embryo complexity increases. There is ubiquitous distribution of LepRs underlying pleiotropism of leptin in all tissues investigated. LepRA1 and LepRA2 are differentially expressed with LepRA1 more abundant than LepRA2 in most of the tissues investigated, with the only exception of liver. Analysis of constitutive LepRA1 and LepRA2 expression in brain and liver at parr, post-smolt and adult stages reveal striking spatial divergence between the duplicates at all stages investigated. This suggests that, beside increased metabolic requirements, leptin sensitivity in the salmon brain might be linked to important variables such as habitat, ecology and life cycle. Furthermore, leptins and LepRs mRNAs in the brain showed gene-specific variability in response to long term fasting, suggesting that leptin's roles as modulator of nutritional status in Atlantic salmon might be governed by distinct genetic evolutionary processes and distinct functions between the paralogs.

Effects of nutritional status on plasma leptin levels and in vitro regulation of adipocyte leptin expression and secretion in rainbow trout.

As leptin has a key role on appetite, knowledge about leptin regulation is important in order to understand the control of energy balance. We aimed to explore the modulatory effects of adiposity on plasma leptin levels in vivo and the role of potential regulators on leptin expression and secretion in rainbow trout adipocytes in vitro. Fish were fed a regular diet twice daily ad libitum or a high-energy diet once daily at two ration levels; satiation (SA group) or restricted (RE group) to 25% of satiation, for 8weeks. RE fish had significantly reduced growth (p<0.001) and adipose tissue weight (p<0.001), and higher plasma leptin levels (p=0.022) compared with SA fish. Moreover, plasma leptin levels negatively correlated with mesenteric fat index (p=0.009). Adipocytes isolated from the different fish were treated with insulin, ghrelin, leucine, eicosapentaenoic acid or left untreated (control). In adipocytes from fish fed regular diet, insulin and ghrelin increased leptin secretion dose-dependently (p=0.002; p=0.033, respectively). Leptin secretion in control adipocytes was significantly higher in RE than in SA fish (p=0.022) in agreement with the in vivo findings, indicating that adipose tissue may contribute to the circulating leptin levels. No treatment effects were observed in adipocytes from the high-energy diet groups, neither in leptin expression nor secretion, except that leptin secretion was significantly reduced by leucine in RE fish adipocytes (p=0.025). Overall, these data show that the regulation of leptin in rainbow trout adipocytes by hormones and nutrients seems to be on secretion, rather than at the transcriptional level.

Neuroendocrine control of appetite in Atlantic halibut (Hippoglossus hippoglossus): changes during metamorphosis and effects of feeding.

Hormones and neuropeptides play a crucial role in the appetite control system of vertebrates, yet few studies have focused on their importance during early teleost development. In this study, we analysed the expression patterns of the appetite-controlling factors ghrelin, neuropeptide Y (NPY), peptide YY (PYY), pro-opiomelanocortin (POMC-C), and cocaine-amphetamine-related transcript (CART) by quantitative PCR. Transcript expression was investigated in response to feeding in developing Atlantic halibut larvae: before (premetamorphic stage 5) and during metamorphosis (stages 8 and 9B), and also in response to a fast-refeed challenge. We show that ghrelin transcript expression increased in synchrony with stomach development, while CART was significantly reduced during larval development. PYY was up-regulated 1 and 3 h after feeding in stage 5. Transcript abundance of other appetite-controlling factors did not change in response to feeding. Fasting-refeeding trials (majority of larvae in metamorphosing stage 7) revealed a down-regulation of POMC-C 30 min after refeeding, while ghrelin, PYY and NPY transcript expression increased 2, 4 and 5 h after refeeding, respectively. In summary, transcripts for key appetite-controlling factors were detected early during development in Atlantic halibut and their emergence was not correlated with metamorphosis, with the exception of ghrelin. Our results suggest that PYY may mediate satiety early in larval development. The differing response times of POMC-C, ghrelin, PYY and NPY to a meal are intriguing and require further exploration to understand the role of each player in appetite control.

Roles of leptin and ghrelin in adipogenesis and lipid metabolism of rainbow trout adipocytes in vitro.

Leptin and ghrelin are important regulators of energy homeostasis in mammals, whereas their physiological roles in fish have not been fully elucidated. In the present study, the effects of leptin and ghrelin on adipogenesis, lipolysis and on expression of lipid metabolism-related genes were examined in rainbow trout adipocytes in vitro. Leptin expression and release increased from preadipocytes to mature adipocytes in culture, but did not affect the process of adipogenesis. While ghrelin and its receptor were identified in cultured differentiated adipocytes, ghrelin did not influence either preadipocyte proliferation or differentiation, indicating that it may have other adipose-related roles. Leptin and ghrelin increased lipolysis in mature freshly isolated adipocytes, but mRNA expression of lipolysis markers was not significantly modified. Leptin significantly suppressed the fatty acid transporter-1 expression, suggesting a decrease in fatty acid uptake and storage, but did not affect expression of any of the lipogenesis or ß-oxidation genes studied. Ghrelin significantly increased the mRNA levels of lipoprotein lipase, fatty acid synthase and peroxisome proliferator-activated receptor-ß, and thus appears to stimulate synthesis of triglycerides as well as their mobilization. Overall, the study indicates that ghrelin, but not leptin seems to be an enhancer of lipid turn-over in adipose tissue of rainbow trout, and this regulation may at least partly be mediated through autocrine/paracrine mechanisms. The mode of action of both hormones needs to be further explored to better understand their roles in regulating adiposity in fish.

Functional modifications associated with gastrointestinal tract organogenesis during metamorphosis in Atlantic halibut (Hippoglossus hippoglossus).

BACKGROUND: Flatfish metamorphosis is a hormone regulated post-embryonic developmental event that transforms a symmetric larva into an asymmetric juvenile. In altricial-gastric teleost fish, differentiation of the stomach takes place after the onset of first feeding, and during metamorphosis dramatic molecular and morphological modifications of the gastrointestinal (GI-) tract occur. Here we present the functional ontogeny of the developing GI-tract from an integrative perspective in the pleuronectiforme Atlantic halibut, and test the hypothesis that the multiple functions of the teleost stomach develop synchronously during metamorphosis. RESULTS: Onset of gastric function was determined with several approaches (anatomical, biochemical, molecular and in vivo observations). In vivo pH analysis in the GI-tract lumen combined with quantitative PCR (qPCR) of α and ß subunits of the gastric proton pump (H+/K+-ATPase) and pepsinogen A2 indicated that gastric proteolytic capacity is established during the climax of metamorphosis. Transcript abundance of ghrelin, a putative orexigenic signalling molecule produced in the developing stomach, correlated (p < 0.05) with the emergence of gastric proteolytic activity, suggesting that the stomach's role in appetite regulation occurs simultaneously with the establishment of proteolytic function. A 3D models series of the GI-tract development indicated a functional pyloric sphincter prior to first feeding. Observations of fed larvae in vivo confirmed that stomach reservoir function was established before metamorphosis, and was thus independent of this event. Mechanical breakdown of food and transportation of chyme through the GI-tract was observed in vivo and resulted from phasic and propagating contractions established well before metamorphosis. The number of contractions in the midgut decreased at metamorphic climax synchronously with establishment of the stomach's proteolytic capacity and its increased peristaltic activity. Putative osmoregulatory competence of the GI-tract, inferred by abundance of Na+/K+-ATPase α transcripts, was already established at the onset of exogenous feeding and was unmodified by metamorphosis. CONCLUSIONS: The functional specialization of the GI-tract was not exclusive to metamorphosis, and its osmoregulatory capacity and reservoir function were established before first feeding. Nonetheless, acid production and the proteolytic capacity of the stomach coincided with metamorphic climax, and also marked the onset of the stomach's involvement in appetite regulation via ghrelin.