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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 (40 d 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. [ABSTRACT FROM PUBLISHER]

A 5-week feeding trial was conducted in the cleaner fish Ballan wrasse ( Labrus bergylta ) for a better understanding of the basic biology of the intestinal functions and health in this stomach less species. During the trial, Ballan wrasse was fed either a reference diet, the reference diet supplemented with (i) a commercial prebiotic (Aquate™ SG, 0·4 %) expected to have beneficial effects, (ii) soya saponins (0·7 %) expected to induce inflammation or (iii) a combination of the prebiotics and the soya saponins to find a remedy for gut inflammation. Blood, intestinal tissue and gut content from four consecutive intestinal segments (IN1 - IN4) were collected. No significant differences in fish growth were observed between the four dietary groups. Saponin supplementation, both alone and in combination with prebiotics, increased weight index of IN2 and IN3 and decreased blood plasma glucose, cholesterol and total protein. Dry matter of intestinal content and activity of digestive enzymes were not affected by diet. Histomorphological analyses revealed a progressing inflammation with increased infiltration by immune cells particularly into the distal parts of the intestine in fish fed diets with saponins, both alone and in combination with prebiotics. Gene expression profiles obtained by RNA sequencing and quantitative PCR mirrored the histological and biochemical changes induced by the saponin load. The study demonstrated that Ballan wrasse gut health and digestive function may be markedly affected by feed ingredients containing antinutrients.

Marine fish larvae often experience high mortality unrelated to predation during early life stages, and farmed ballan wrasse ( Labrus bergylta ) is no exception. Knowing when the adaptive immune system is developed and fully functional, and how nutrition may modulate these processes is therefore of importance to establish effective prophylactic measures and will also extend the relatively limited knowledge on the immune system in lower vertebrates. The thymus anlage of ballan wrasse was found to be histologically visible for the first time at larval stage 3 (20-30 days post hatch, dph) and becomes lymphoid at stage 5 (50-60 dph) correlating with an increase of T-cell marker transcripts. At this stage, a clear zonation into a RAG1 + cortex and a RAG1 - CD3ϵ + medulla was distinguished, indicating that T-cell maturation processes in ballan wrasse are similar to other teleosts. The higher abundance of CD4-1 + compared to CD8β + cells in the thymus together with the apparent lack of CD8β + cells in gill, gut, and pharynx, where CD4-1 + cells were identified, indicates that helper T-cells have a more prominent role during larval development compared to cytotoxic T-cells. As ballan wrasse lacks a stomach but has an exceptionally high IgM expression in the hindgut, we hypothesize that helper T-cells are crucial for activation and recruitment of IgM + B-cells and possibly other leukocytes to the gut during early development. Nutritional factors such as DHA/EPA, Zn and Se may lead to an earlier expression of certain T-cell markers as well as a larger size of the thymus, indicating an earlier onset of adaptive immunity. Including live feeds that supplies the larva with higher amounts of these nutrients can therefore be beneficial for ballan wrasse farming., 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 Etayo, Lie, Bjelland, Hordvik, Øvergård and Sæle.)

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.)

Serotonin (5-HT) is pivotal in the complex regulation of gut motility and consequent digestion of nutrients via multiple receptors. We investigated the serotonergic system in an agastric fish species, the ballan wrasse (Labrus bergylta) as it represents a unique model for intestinal function. Here we present evidence of the presence of enterochromaffin cells (EC cells) in the gut of ballan wrasse comprising transcriptomic data on EC markers like adra2a, trpa1, adgrg4, lmxa1, spack1, serpina10 , as well as the localization of 5-HT and mRNA of the rate limiting enzyme; tryptophan hydroxylase ( tph1 ) in the gut epithelium. Second, we examined the effects of dietary marine lipids on the enteric serotonergic system in this stomach-less teleost by administrating a hydrolyzed lipid bolus in ex vivo guts in an organ bath system. Modulation of the mRNA expression from the tryptophan hydroxylase tph1 (EC cells isoform), tph2 (neural isoform), and other genes involved in the serotonergic machinery were tracked. Our results showed no evidence to confirm that the dietary lipid meal did boost the production of 5-HT within the EC cells as mRNA tph1 was weakly regulated postprandially. However, dietary lipid seemed to upregulate the post-prandial expression of tph2 found in the serotonergic neurons. 5-HT in the intestinal tissue increased 3 hours after "exposure" of lipids, as was observed in the mRNA expression of tph2 . This suggest that serotonergic neurons and not EC cells are responsible for the substantial increment of 5-HT after a lipid-reach "meal" in ballan wrasse. Cells expressing tph1 were identified in the gut epithelium, characteristic for EC cells. However, Tph1 positive cells were also present in the lamina propria. Characterization of these cells together with their implications in the serotonergic system will contribute to broad the scarce knowledge of the serotonergic system across teleosts., 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 Etayo, Le, Araujo, Lie and Sæle.)

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.

Cholecystokinin (CCK) is well-known as a key hormone that inhibits stomach emptying and stimulates midgut motility in gastric species. However, the function of CCK related to gut motility in agastric fish, especially in fish with a short digestive tract such as ballan wrasse, remains unknown. Here we present a detailed description of the spatio-temporal quantification of intestinal motility activity in vitro comprising the complete intestinal tract in ballan wrasse. We show that CCK modulates intestinal motility, having multiple effects on motility patterns depending on location in the gut and types of contractions. CCK reduced propagating contractions in the foregut, but it increased both non-propagating and propagating contractions in the hindgut. CCK also altered the direction of propagating contractions, as it reduced anterograde ripples and slow propagating contractions. The velocity of propagating contractions was slowed down by CCK. CCK also reduced the amplitude of standing contractions and ripples, but it did not alter the amplitude of slow propagating contractions. The presence of CCKA receptor antagonist modulated the motility responses of ballan wrasse intestines when exposed to CCK. We also showed that CCK reduced the intestinal length and stimulated motility to empty the gallbladder. Based on our findings we hypothesize that CCK, mainly through the CCKA receptor, modulates non-propagating and propagating contractions to optimize digestion and absorption and regulate the intestinal evacuation in ballan wrasse. We also found evidence that the modulation of intestinal motility by CCK is different in agastric fish from that in gastric vertebrates. We suggest that this is an evolutionary adaptation to optimize digestion without a stomach.

Previously, somatic hypermutation (SHM) was considered to be exclusively associated with affinity maturation of antibodies, although it also occurred in T cells under certain conditions. More recently, it has been shown that SHM generates diversity in the variable domain of T cell receptor (TCR) in camel and shark. Here, we report somatic mutations in TCR alpha chain genes of the teleost fish, Ballan wrasse ( Labrus bergylta ), and show that this mechanism adds extra diversity to the polymorphic constant (C) region as well. The organization of the TCR alpha/delta locus in Ballan wrasse was obtained from a scaffold covering a single copy C alpha gene, 65 putative J alpha segments, a single copy C delta gene, 1 J delta segment, and 2 D delta segments. Analysis of 37 fish revealed 6 allotypes of the C alpha gene, each with 1-3 replacement substitutions. Somatic mutations were analyzed by molecular cloning of TCR alpha chain cDNA. Initially, 79 unique clones comprising four families of variable (V) alpha genes were characterized. Subsequently, a more restricted PCR was performed to focus on a specific V gene. Comparison of 48 clones indicated that the frequency of somatic mutations in the VJ region was 4.5/1,000 base pairs (bps), and most prevalent in complementary determining region 2 (CDR2). In total, 45 different J segments were identified among the 127 cDNA clones, counting for most of the CDR3 diversity. The number of mutations in the C alpha chain gene was 1.76 mutations/1,000 bps and A nucleotides were most frequently targeted, in contrast to the VJ region, where G nucleotides appeared to be mutational hotspots. The replacement/synonymous ratios in the VJ and C regions were 2.5 and 1.85, respectively. Only 7% of the mutations were found to be linked to the activation-induced cytidine deaminase hotspot motif (RGYW/WRCY).

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.

Selenium (Se) and its derivatives are known to have protective effects against mercury (Hg) toxicity in mammals. In this study we wanted to evaluate whether Se co-exposure affect the transcription of methylmercury (MeHg) toxicity-relevant genes in early life stages of fish. Juvenile Atlantic cod were exposed to regular feed (control), Se-spiked feed (3mg Se kg(-1)), MeHg-spiked feed (10mg Hg kg(-1)) or to Se- and MeHg-spiked feed (3mg Se kg(-1) and 10mg Hg kg(-1), respectively) for ten weeks. Liver tissue was harvested for transcriptional analysis when the fish were weighing 11.4 ± 3.2g. Accumulated levels of Hg in liver of the two groups of fish exposed to MeHg were 1.5mg Hg kg(-1) wet weight, or 44-fold higher than in the control group, while the Se concentrations differed with less than 2-fold between the fish groups. Selenium co-exposure had no effect on the accumulated levels of Hg in liver tissue; however, MeHg co-exposure reduced the accumulated level of Se. Dietary exposure to MeHg had no effect on fish growth. Interaction effects between Se and MeHg exposure were observed for the transcriptional levels of CAT, GPX1, GPX3, NFE2L2, UBA52, SEPP1 and DNMT1. Significant effects of MeHg exposure were seen for DNMT1 and PPARG, while effects of Se exposure were seen for GPX4B and SEPP1A, as well as for DNA methyltransferase activity. The transcriptional results suggest, by considering up-regulation as a proxy for negative impact and at the tested concentrations, a pro-oxidative effect of Se co-exposure with MeHg, rather than an antioxidative effect., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We have previously described that fish larvae absorb a larger fraction of dietary monoacylglycerol than TAG. To investigate how dietary hydrolysed lipids affect a vertebrate at early life stages over time, we fed Atlantic cod (Gadus morhua) larvae six diets with different degrees of hydrolysed lipids for 30 d. The different diets had no effect on growth, but there was a positive correlation between the level of hydrolysed lipids in the diets and mortality. Important genes in lipid metabolism, such as PPAR, farnesoid X receptor (FXR) and stearoyl-CoA desaturase (SCD), were regulated by the different diets. Genes involved in the oxidative stress response did not respond to the increased lipid hydrolysation in the diets. However, enterocyte damage was observed in animals fed diets with 2.7 % NEFA (diet 3) or more. It is thus possible that mortality was due to infections and/or osmotic stress due to the exposure of the subepithelial tissue. In contrast to earlier experiments showing a positive effect of dietary hydrolysed lipids, we have demonstrated a toxic effect of dietary NEFA on Atlantic cod larvae. Toxicity is not acute but needs time to accumulate.

The pancreatic enzyme secretory phospholipase A2 group IB (sPLA2 IB) hydrolyses phospholipids at the sn-2 position, resulting in a NEFA and a lyso-phospholipid, which are then absorbed by the enterocytes. The sPLA2 IB is a member of a family of nineteen enzymes sharing the same catalytic ability, of which nine are cytosolic and ten are secretory. Presently, there are no pharmacological tools to separate between the different secretory enzymes when measuring the enzymatic activity. Thus, it is important to support activity data with more precise techniques when isolation of intestinal content is not possible for analysis, as in the case of small teleost larvae, where the whole animal is sometimes analysed. In the present study, we characterise the sPLA2 IB gene in Atlantic cod (Gadus morhua) and describe its ontogeny at the genetic and protein level and compare this to the total sPLA2 activity level. A positive correlation was found between the expression of sPLA2 IB mRNA and protein. Both remained stable and low during the larval stage followed by an increase from day 62 posthatch, coinciding with the development of the pyloric ceaca. Meanwhile, total sPLA2 enzyme activity in cod was stable and relatively high during the early stages when larvae were fed live prey, followed by a decrease in activity when the fish were weaned to a formulated diet. Thus, the expression of sPLA2 IB mRNA and protein did not correlate with total sPLA2 activity.

Background: The aim of this work was to study how evenly detoxifying genes are transcribed spatially in liver tissue of fish. Ten Atlantic salmon Salmo salar were intraperitoneally injected with 50 mg/kg of the strong CYP1A inducer beta-naphthoflavone and liver tissue harvested seven days later. The liver from 10 control and 10 exposed fish were split into eight sections, RNA extracted and three reference (beta-actin, elongation factor 1AB (EF1AB)) and two detoxifying genes (CYP1A and GST) quantified with real-time RT-PCR. The cellular localization of the EF1AB and CYP1A mRNA in the liver of control and beta-naphthoflavone treated fish was then determined by in situ hybridization (ISH) using EF1AB and CYP1A biotinylated oligonucleotide probes., Results: The study shows that genes encoding phase I and phase II conjugating enzymes are unevenly transcribed in different parts of the liver of Atlantic salmon seven days after a single-dose of beta-naphthoflavone exposure. Transcription of CYP1A and GST was higher in the middle section of the liver compared to the distal and proximal parts of the organ. The ISH data suggest that CYP1A transcription happens mainly in hepatocyte cells in the liver, and that hepatocytes in the vicinity of blood vessels respond stronger to beta-naphthoflavone than cells further away from the blood supply., Conclusion: Overall, the qRT-PCR and ISH results reported here suggest that gene expression analysis should be performed on as pure cell populations as possible. If bulk tissue samples are to be used, one should always check how evenly the target genes are expressed in tissue sections and organs in every study.