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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.)

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., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Similar to many marine teleost species, Atlantic cod undergo remarkable physiological changes during the early life stages with concurrent and profound changes in feeding biology and ecology. In contrast to the digestive system, very little is known about the ontogeny and the localization of the centers that control appetite and feed ingestion in the developing brain of fish. We examined the expression patterns of three appetite regulating factors (orexigenic: neuropeptide Y, NPY; prepro-orexin, pOX and anorexigenic: cocaine- and amphetamine-regulated transcript, CART) in discrete brain regions of developing Atlantic cod using chromogenic and double fluorescent in situ hybridization. Differential temporal and spatial expression patterns for each appetite regulator were found from first feeding (4 days post hatch; dph) to juvenile stage (76 dph). Neurons expressing NPY mRNA were detected in the telencephalon (highest expression), diencephalon, and optic tectum from 4 dph onward. CART mRNA expression had a wider distribution along the anterior-posterior brain axis, including both telencephalon and diencephalon from 4 dph. From 46 dph, CART transcripts were also detected in the olfactory bulb, region of the nucleus of medial longitudinal fascicle, optic tectum and midbrain tegmentum. At 4 and 20 dph, pOX mRNA expression was exclusively found in the preoptic region, but extended to the hypothalamus at 46 and 76 dph. Co-expression of both CART and pOX genes were also observed in several hypothalamic neurons throughout larval development. Our results show that both orexigenic and anorexigenic factors are present in the telencephalon, diencephalon and mesencephalon in cod larvae. The telencephalon mostly contains key factors of hunger control (NPY), while the diencephalon, and particularly the hypothalamus may have a more complex role in modulating the multifunctional control of appetite in this species. As the larvae develop, the overall progression in temporal and spatial complexity of NPY, CART and pOX mRNAs expression might be correlated to the maturation of appetite control regulation. These observations suggest that teleost larvae continue to develop the regulatory networks underlying appetite control after onset of exogenous feeding.

The growth and development of marine fish larvae fed copepods is superior to those fed rotifers, but the underlying molecular reasons for this are unclear. In the following study we compared the effects of such diets on redox regulation pathways during development of Atlantic cod (Gadus morhua) larvae. Cod larvae were fed a control diet of copepods or the typical rotifer/Artemia diet commonly used in commercial marine fish hatcheries, from first feeding until after metamorphosis. The oxidised and reduced glutathione levels, the redox potential, and the mRNA expression of 100 genes in redox system pathways were then compared between treatments during larval development. We found that rotifer/Artemia-fed cod larvae had lower levels of oxidised glutathione, a more reduced redox potential, and altered expression of approximately half of the redox system genes when compared to copepod-fed larvae. This rotifer/Artemia diet-induced differential regulation of the redox system was greatest during periods of suboptimal growth. Upregulation of the oxidative stress response transcription factor, nrf2, and NRF2 target genes in rotifer/Artemia fed larvae suggest this diet induced an NRF2-mediated oxidative stress response. Overall, the data demonstrate that nutritional intake plays a role in regulating the redox system in developing fish larvae. This may be a factor in dietary-induced differences observed in larval growth., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

In order to maximize protein digestion, the release of enzymes into the gut lumen is closely controlled by a regulatory loop. Cholecystokinin (CCK) is among the enteric hormones that play a key role in the control of digestive enzyme secretion, but its role in first-feeding larvae is still unclear and may differ between species. However, in all marine fish larvae that have not developed a stomach by first-feeding, trypsin is the most important proteolytic enzyme. In order to examine the regulation and feedback mechanisms in the gut of larval cod, we therefore studied the interactions between cholecystokinin and tryptic enzyme activity following the administration of solutions containing test substances directly into the gut. We tube-fed a single dose of physiological saline solution containing either CCK, CCK antagonist, trypsin inhibitor, phytohemagglutinin (PHA; a possible trigger for the digestive response) or physiological saline alone, while a further control group was left untreated. We then followed the response in CCK and tryptic enzyme activity for 0.5-8h after the administration. We performed the experiment on larvae at 26day post first-feeding, which is before the stomach has evolved and the size of the larvae allows easier handling. Individual larvae were analyzed for CCK and tryptic enzyme activity using radioimmunoassay and fluorimetric techniques respectively. Both factors varied over time in the untreated control group, possibly due to an endogenous daily rhythm. The higher CCK levels at 4h and 8h in the saline-injected group may be caused by reflexes initiated by distension of the gut. An increase in tryptic enzyme activity after injection of CCK supports the hypothesis that this hormone plays a part in the release of pancreatic enzymes in larval cod at this developmental stage. However, administration of a CCK antagonist and a trypsin inhibitor did not reveal conclusive results, probably due to the relatively low concentrations used. The response in tryptic activity in the PHA group was similar to the administration of CCK, pointing towards a stimulatory effect of PHA on the proteolytic enzyme capacity of cod larvae., (© 2013.)

The hypothesis of the present study was that cod larvae have a limitation in lipid digestion, and that absorption of lipids would increase by pre-hydrolysation. The diets used were designed to contain 15% lipid, of which 40% was phosphatidylcholine (PC) and 60 % was TAG. Cod larvae (40d post hatch (dph)) were fed a single meal where either PC or TAG was radioactively labelled, and the labelled PC or TAG was either intact or hydrolysed (pre-digested). The larvae were then incubated individually in chambers with collection of CO2 for 10 h. The following fractions were analysed for radioactivity: the incubation water (evacuated feed); the intestine; the body; the CO2 trap. The larvae ate a 16-29 μg diet, equivalent to 3·4-5·2 % of dry body weight. In the whole population, 0-16% of the lipid was evacuated. The larvae that had eaten less than 1·9-2·7 μg lipid absorbed close to 100% of the lipid, absorption being defined conservatively as the amount contained in the carcass and CO2, excluding the intestinal tissue. In these larvae, approximately 100 % of the absorbed lipid was also catabolised. In the larvae that ingested more than 1·9-2·7 μg lipid, there was a linear reduction in lipid absorption to a minimum of 55% at the highest lipid intakes parallel to an increasing retention of lipids in the carcass. There were only minor differences in digestion, absorption, retention and metabolism of lipids between the larvae fed the different diets, and the larvae tended to retain lipid classes as they were present in the feed. The study shows that 40-dph Atlantic cod larvae have an efficient utilisation of dietary lipids supplied as intact PC and TAG.

The effects of dietary inclusions of size-fractionated peptides and free amino acids (FAAs) on Peptide Transporter 1 (PepT1) mRNA levels were assessed along the length of the intestine of juvenile Atlantic cod (Gadus morhua). Five groups of fish (10-15g) were fed for 46days on diets containing approximately 42% protein, provided either as fish meal (FM, control diet) or as a combination of FM with whole fish hydrolysate (FH), retenate after ultrafiltration of FH (UFR), nanofiltered retenate of FH (NFR), or a mix of FAAs, at a 30% level of FM substitution. PepT1 mRNA expression was assessed in pyloric caeca (S1) and the remainder of the intestine divided into four equally long segments (S2-S5). PepT1 transcripts were found in all segments, indicating that the whole intestine is involved in peptide absorption. Differences in the regional expression profile of PepT1 were found. Under control diet (FM diet) conditions, fish exhibited a reduced expression in S5 compared to S2. In fish fed FAA and UFR diets, PepT1 mRNA levels were higher in S2 and S3 compared to other regions. These data suggest that PepT1 may be variably recruited along the whole intestine, including the most distal part, in response to changes in the luminal protein source content. This adaptive response might be functional to keep a maximal efficiency of protein absorption at the intestinal level., (2010 Elsevier Inc. All rights reserved.)

One of the main gastrointestinal hormones, cholecystokinin (CCK), was studied in order to advance understanding of the control of the digestive process in Atlantic cod Gadus morhua larvae after onset of first feeding. Larvae were fed either natural zooplankton or enriched rotifers in similar rearing systems and sampled from hatching to 22 days post-hatch (dph). CCK was visualized by immunohistochemistry and the first CCK-immunoreactive (IR) cells were detected at 8 dph corresponding to 6 days after first feeding. The CCK-IR cells were mostly found in the anterior midgut, and the number of CCK-IR cells was lower in the posterior midgut. They were also present in the hindgut of some of the larvae, but not in the foregut. No clear differences were found in the ontogenetic appearance and the distribution pattern of CCK-IR cells between the two dietary treatments. This indicates that the onset of CCK production in the gut as well as the spatial distribution of the CCK-IR cells is not differentially affected by these diets. To what extent the hormone production itself is influenced by dietary factors needs to be studied by more sensitive methods.

The intestinal absorption of di- and tri-peptides generally occurs via the oligopeptide transporter, PepT1. This study evaluates the expression of PepT1 in larval Atlantic cod (Gadus morhua) during the three weeks following the onset of exogenous feeding. Larval Atlantic cod were fed either wild captured zooplankton or enriched rotifers. cDNA was prepared from whole cod larvae preceding first feeding and at 1000 each Tuesday and Thursday for the following three weeks. Spatial and temporal expression patterns of PepT1 mRNA were compared between fish consuming the two prey types using in situ hybridization and quantitative real-time PCR. Results indicated that PepT1 mRNA was expressed prior to the onset of exogenous feeding. In addition, PepT1 was expressed throughout the digestive system except the esophagus and sphincter regions. Expression slightly increased following first-feeding and continued to increase throughout the study for larvae feeding on both prey types. When comparing PepT1 expression in larvae larger than 0.15-mg dry mass with expression levels in larvae prior to feeding, no differences were detected for larvae fed rotifers, but the larvae fed zooplankton had significantly greater PepT1 expression at the larger size. In addition, PepT1 expression in the zooplankton fed larvae larger than 0.15-mg dry mass had significantly greater expression than rotifer fed larvae of a similar weight. Switching prey types did not affect PepT1 expression. These results indicate that Atlantic cod PepT1 expression was slightly different relative to dietary treatment during the three weeks following first-feeding. In addition, PepT1 may play an important role in the larval nutrition since it is widely expressed in the digestive tract.

A novel full-length cDNA that encodes for the Atlantic cod (Gadus morhua L.) PepT1-type oligopeptide transporter has been cloned. This cDNA (named codPepT1) was 2,838 bp long, with an open reading frame of 2,190 bp encoding a putative protein of 729 amino acids. Comparison of the predicted Atlantic cod PepT1 protein with zebrafish, bird and mammalian orthologs allowed detection of many structural features that are highly conserved among all the vertebrate proteins analysed, including (1) a larger than expected area of hydrophobic amino acids in close proximity to the N terminus; (2) a single highly conserved cAMP/cGMP-dependent protein kinase phosphorylation motif; (3) a large N-glycosylation-rich region within the large extracellular loop; and (4) a conserved and previously undescribed stretch of 8-12 amino acid residues within the large extracellular loop. Expression analysis at the mRNA level indicated that Atlantic cod PepT1 is mainly expressed at intestinal level, but that it is also present in kidney and spleen. Analysis of its regional distribution along the intestinal tract of the fish revealed that PepT1 is ubiquitously expressed in all segments beyond the stomach, including the pyloric caeca, and through the whole midgut. Only in the last segment, which included the hindgut, was there a lower expression. Atlantic cod PepT1, the second teleost fish PepT1-type transporter documented to date, will contribute to the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful tool for the study of gut functional regionalization, as well as a marker for the analysis of temporal and spatial expression during ontogeny.