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.publishedVersio

Spatial transcription of CYP1A in fish liver

<p>Abstract</p> <p>Background</p> <p>The aim of this work was to study how evenly detoxifying genes are transcribed spatially in liver tissue of fish. Ten Atlantic salmon <it>Salmo salar </it>were intraperitoneally injected with 50 mg/kg of the strong CYP1A inducer β-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 (β-actin, elongation factor 1A_B(EF1A_B)) and two detoxifying genes (CYP1A and GST) quantified with real-time RT-PCR. The cellular localization of the EF1A_Band CYP1A mRNA in the liver of control and β-naphthoflavone treated fish was then determined by <it>in situ </it>hybridization (ISH) using EF1A_Band CYP1A biotinylated oligonucleotide probes.</p> <p>Results</p> <p>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 β-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 β-naphthoflavone than cells further away from the blood supply.</p> <p>Conclusion</p> <p>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.</p

Physical and nutrient stimuli differentially modulate gut motility patterns, gut transit rate, and transcriptome in an agastric fish, the ballan wrasse

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.publishedVersio

The thymus and T-cell ontogeny in ballan wrasse (Labrus bergylta) is nutritionally modelled

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.publishedVersio

Effects of cholecystokinin (CCK) on gut motility in the stomachless fish ballan wrasse (Labrus bergylta)

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.publishedVersio

Effect of dietary electrolyte balance on the interplay between water fluxes and digestive functioning along the gastrointestinal tract of freshwater rainbow trout (Oncorhynchus mykiss)

Aquaculture feed formulation is shifting from fish meal and fish oil toward other ingredients, such as plant-based ingredients, which lead to different levels and forms of minerals in diets. Dietary minerals are essential not only for growth, but also for acid-base balance and the homeostasis of fish body fluids. The gastrointestinal tract (GIT) is involved in the process of osmoregulation of salt and water during digestion, but this process can be hampered when the dietary electrolyte balance (dEB = Na - K + Cl, mEq/kg) is altered. To better understand the interaction between feeding and osmoregulation along the GIT, two diets with low (−100 mEq/kg) and high (+600 mEq/kg) dEB were designed. Freshwater rainbow trout (average initial weight, 306 g) were fed for 6 weeks. Faeces were collected during the last week for digestibility analyses. The final sampling took place over three days, with fish being sampled at 2 time points after feeding (3 and 7 h) and dissected to collect chyme from 4 GIT segments: stomach, proximal, middle and distal intestine. Chyme was analysed for dry matter, pH, osmolality, crude protein (CP) and mineral content. Yttrium oxide (Y2O3) was used as an inert marker to measure water fluxes, mineral fluxes, and nutrient digestibility from different gut segments. Both dEB and time after feeding altered (p 0.05). dEB also affected (p < 0.01) chyme pH and sodium (Na) fluxes in the stomach. Faecal digestibility of dry matter (DM) and CP was higher (p < 0.001) when fish were fed a high dEB diet compared to a low dEB diet. The opposite was observed for Ca availability, which was lower at the high dEB diet than at the low dEB diet (p < 0.001). In contrast to faecal digestibility, CP and mineral digestibility measured in the different gut segments were different predominantly in the middle and distal intestine. Our findings suggest that the GIT plays a role in regulating the alterations caused by the contrasting dEB, and that this regulation is stronger in the middle and distal intestine. Furthermore, this regulation affects nutrient and mineral digestibility in the middle and distal segments of the GIT.publishedVersio

Optimization of the Balance between Protein, Lipid and Carbohydrate in Diets for Lumpfish (Cyclopterus lumpus)

The aim of this study was to investigate how the dietary balance between protein, lipid, and carbohydrate affects growth, welfare, and health with focus on immune responses, in lumpfish of body weight ranges 1.7-10 g and 15-50 g. A three-component mixture design, with simultaneous variation of the three macronutrients was applied. Growth, tissue and plasma nutrient composition, welfare, cataract frequency, and immune responses in leukocytes isolated from the fish head kidney were studied. Most responses were linear and driven by dietary lipid level, with minor effects of carbohydrate and protein. The growth in 1.7-10 g fish followed a special cubic model with no clear optimum. The fastest growth in the 15-50 g fish was obtained with the diet containing 55% protein, 17% fat, and 6% carbohydrate. The high dietary levels of lipid also gave high lipid levels in the tissues and a trend of improved welfare score, but there were no effects on cataract. The diets with minimum and maximum protein gave suboptimal immune responses in isolated head kidney cells of the 15-50 g fish, while the fastest growing fish showed normal immune responses. We propose that diets for lumpfish from 10-50 g body weight should contain approximately 55% protein, minimum 10% lipid, and maximum 10% carbohydrate. These diets will also be suitable for lumpfish of 1.7-10 g.publishedVersio

Ossification of Atlantic cod (Gadus morhua) – Developmental stages revisited

In studies of marine larvae, it is common to use days post-hatch as a developmental reference point. We show that age is a poor measure of morphological and physiological development in Atlantic cod. Therefore, we propose a set of five developmental stages of Atlantic cod fromstart-feeding until the juvenile stage, based on cranial ossification as previously done in Atlantic halibut. Cod follows a sequence of cranial ossification that is to a large extent preserved in most fish species examined. These stages are therefore tools to standardize sampling and to reduce growth dependent variation in the analysis of larvae during development. Weshow that several developmental stages are present in the same rearing unit at a given time. We also demonstrate that nutrition during early development is a vital foundation for robust skeletal development. Cod larvae supplied with copepods instead of rotifers followed by Artemia, develop less skeletal deformities at 10 cm standard length, despite given the same formulated feed from 1.8 cm standard length and onwards. Statement of relevance: This paper provides developmental stages that are vital for best practice protocols in aquaculture. By relating farming practices to developmental stages and not age, the right treatment ect may be provided. This manuscript does also highlight the importance of nutrition during live feed stages on events that may occur late in the production cycle.acceptedVersio

Intracellular trafficking of fatty acids in the fish intestinal epithelial cell line RTgutGC

The shift towards higher inclusion of vegetable oils (VOs) in aquafeeds has resulted in major changes in dietary fatty acid composition, especially increased amounts of monounsaturated fatty acids (MUFAs) and decreased polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SFAs). However, little is known about how this change in fatty acid (FA) profile affects the intracellular fate of these fatty acids in the intestinal cells. To investigate this topic, we used the rainbow trout intestinal epithelial cell line (RTgutGC) as an in vitro model. The cells were incubated with either palmitic acid (16:0, PA), oleic acid (18:1n-9, OA), or arachidonic acid (20:4n-6, ARA), to represent the SFA, MUFA, and PUFA, respectively. In all experiments, the RTgutGC were incubated with either non-labeled or radiolabeled FA (PA, OA, or ARA) for 16 h at 190C. The cells were then analyzed for the occurrence of cytosolic lipid droplets (CLD) with confocal microscopy, transcriptomic analysis (non-labeled FA experiments) and lipid class composition in the cells and serosal media from the basolateral side of the cells (radiolabeled FA experiments). CLD accumulation was higher in RTgutGC exposed to OA compared to cells given PA or ARA. This was coupled with increased volume, diameter, and surface area of CLDs in OA treated cells than with other FAs (PA, ARA). The results from radiolabeled FAs performed on permeable transwell inserts showed that OA increased the triacylglycerides (TAG) synthesis and was primarily stored in the cells in CLDs; whereas a significant amount of ARA was transported as TAG to the basolateral compartment. A significant proportion of free FAs was found to be excreted to the serosal basolateral side by the cells, which was significantly higher for PA and OA than ARA. Although there were clear clusters in differentially expressed genes (DEGs) for each treatment group, results from transcriptomics did not correlate to lipid transport and CLD analysis. Overall, the accumulation of TAG in CLDs was higher for oleic acid (OA) compared to arachidonic acid (ARA) and palmitic acid (PA). To conclude, carbon chain length and saturation level of FA differently regulate their intracellular fate during fatty acid absorption

Technical feed quality influences health, digestion patterns, body mineralization and bone development in farming of the stomachless cleaner fish ballan wrasse (Labrus bergylta)

Farmed ballan wrasse (Labrus bergylta) is an efficient cleaner fish used for non-medicinal delicing of Atlantic salmon in sea cages replacing to an increasing degree wild wrasse due to considerations for biodiversity and risk of overfishing local wrasse populations. Farming of ballan wrasse has been hampered by low growth rates, high prevalence of skeletal deformities and other welfare related pathologies. In this study we investigated how diets identical in composition but differing in their technical characteristics, by being prepared using different feed production technologies, affect fish performance, mineralization, bone development and gut health of the ballan wrasse larvae and juveniles. The different production technologies include the commonly used ‘high temperature’ extrusion, cold extrusion, and agglomeration, resulting in feed pellets with distinctive physicochemical properties. The results revealed that prolonged feeding periods with extruded pellets during ballan wrasse larvae weaning result in low body mineralization and the development of severe skeletal deformities. In juvenile ballan wrasse, the extruded pellet treatment resulted in higher mortality rates, fish with larger livers, indication for increased serum TAG and cholesterol in a similar manner, and increased activity of the digestive enzymes LAP and maltase, most probably as a compensatory mechanism to the assumed reduced availability of protein and carbohydrates of extruded pellets for this fish species. Smaller dietary effects were identified in terms of intestinal morphology and gene transcription rates.publishedVersio