Heterochiasmy and the establishment of gsdf as a novel sex determining gene in Atlantic halibut.

Atlantic Halibut (Hippoglossus hippoglossus) has a X/Y genetic sex determination system, but the sex determining factor is not known. We produced a high-quality genome assembly from a male and identified parts of chromosome 13 as the Y chromosome due to sequence divergence between sexes and segregation of sex genotypes in pedigrees. Linkage analysis revealed that all chromosomes exhibit heterochiasmy, i.e. male-only and female-only meiotic recombination regions (MRR/FRR). We show that FRR/MRR intervals differ in nucleotide diversity and repeat class content and that this is true also for other Pleuronectidae species. We further show that remnants of a Gypsy-like transposable element insertion on chr13 promotes early male specific expression of gonadal somatic cell derived factor (gsdf). Less than 4.5 MYA, this male-determining element evolved on an autosomal FRR segment featuring pre-existing male meiotic recombination barriers, thereby creating a Y chromosome. Our findings indicate that heterochiasmy may facilitate the evolution of genetic sex determination systems relying on linkage of sexually antagonistic loci to a sex-determining factor.

Quantitative proteome profiling reveals molecular hallmarks of egg quality in Atlantic halibut: impairments of transcription and protein folding impede protein and energy homeostasis during early development.

BACKGROUND: Tandem mass tag spectrometry (TMT labeling-LC-MS/MS) was utilized to examine the global proteomes of Atlantic halibut eggs at the 1-cell-stage post fertilization. Comparisons were made between eggs judged to be of good quality (GQ) versus poor quality (BQ) as evidenced by their subsequent rates of survival for 12 days. Altered abundance of selected proteins in BQ eggs was confirmed by parallel reaction monitoring spectrometry (PRM-LC-MS/MS). Correspondence of protein levels to expression of related gene transcripts was examined via qPCR. Potential mitochondrial differences between GQ and BQ eggs were assessed by transmission electron microscopy (TEM) and measurements of mitochondrial DNA (mtDNA) levels. RESULTS: A total of 115 proteins were found to be differentially abundant between GQ and BQ eggs. Frequency distributions of these proteins indicated higher protein folding activity in GQ eggs compared to higher transcription and protein degradation activities in BQ eggs. BQ eggs were also significantly enriched with proteins related to mitochondrial structure and biogenesis. Quantitative differences in abundance of several proteins with parallel differences in their transcript levels were confirmed in egg samples obtained over three consecutive reproductive seasons. The observed disparities in global proteome profiles suggest impairment of protein and energy homeostasis related to unfolded protein response and mitochondrial stress in BQ eggs. TEM revealed BQ eggs to contain significantly higher numbers of mitochondria, but differences in corresponding genomic mtDNA (mt-nd5 and mt-atp6) levels were not significant. Mitochondria from BQ eggs were significantly smaller with a more irregular shape and a higher number of cristae than those from GQ eggs. CONCLUSION: The results of this study indicate that BQ Atlantic halibut eggs are impaired at both transcription and translation levels leading to endoplasmic reticulum and mitochondrial disorders. Observation of these irregularities over three consecutive reproductive seasons in BQ eggs from females of diverse background, age and reproductive experience indicates that they are a hallmark of poor egg quality. Additional research is needed to discover when in oogenesis and under what circumstances these defects may arise. The prevalence of this suite of markers in BQ eggs of diverse vertebrate species also begs investigation.

Structural and functional maturation of skin during metamorphosis in the Atlantic halibut (Hippoglossus hippoglossus).

To establish if the developmental changes in the primary barrier and osmoregulatory capacity of Atlantic halibut skin are modified during metamorphosis, histological, histochemical, gene expression and electrophysiological measurements were made. The morphology of the ocular and abocular skin started to diverge during the metamorphic climax and ocular skin appeared thicker and more stratified. Neutral mucins were the main glycoproteins produced by the goblet cells in skin during metamorphosis. Moreover, the number of goblet cells producing neutral mucins increased during metamorphosis and asymmetry in their abundance was observed between ocular and abocular skin. The increase in goblet cell number and their asymmetric abundance in skin was concomitant with the period that thyroid hormones (THs) increase and suggests that they may be under the control of these hormones. Several mucin transcripts were identified in metamorphosing halibut transcriptomes and Muc18 and Muc5AC were characteristic of the body skin. Na+, K+-ATPase positive (NKA) cells were observed in skin of all metamorphic stages but their number significantly decreased with the onset of metamorphosis. No asymmetry was observed between ocular and abocular skin in NKA cells. The morphological changes observed were linked to modified skin barrier function as revealed by modifications in its electrophysiological properties. However, the maturation of the skin functional characteristics preceded structural maturation and occurred at stage 8 prior to the metamorphic climax. Treatment of Atlantic halibut with the THs disrupter methimazole (MMI) affected the number of goblet cells producing neutral mucins and the NKA cells. The present study reveals that the asymmetric development of the skin in Atlantic halibut is TH sensitive and is associated with metamorphosis and that this barrier's functional properties mature earlier and are independent of metamorphosis.

Vitamin A and arachidonic acid altered the skeletal mineralization in Atlantic cod (Gadus morhua) larvae without any interactions on the transcriptional level.

The main object of this study was to evaluate the impact of different levels of vitamin A (VA) and arachidonic acid (ARA) in relation to eicosapentaenoic acid (EPA) on mineralization and gene expression in Atlantic cod larvae (Gadus morhua). First-feeding larvae were fed enriched rotifers from start-feeding until 29 days post hatch (dph). Larvae in four tanks were fed one of the following diets: control (EPA/ARA ratio: 15.8, 0.9µg VA g(-1)), control+VA (EPA/ARA ratio: 15.8, 7.8µg VA g(-1)), High ARA (EPA/ARA ratio: 0.9, 1.5µg VA g(-1)) or High ARA+VA (EPA/ARA ratio: 0.9, 12.0µg VA g(-1)). Larvae fed High ARA+VA were shorter at 29dph compared to the other groups and had significantly less mineralized bones when comparing larvae of similar size, showing interaction effects between VA and ARA. Although transcriptomic analysis did not reveal any interaction effects, a higher number of genes were differentially expressed in the high ARA fed larvae compared to control+VA fed larvae. Furthermore, bglap1, bglap2 and col10a1 were all down-regulated in larvae fed High ARA-diets and to a greater extent than larvae fed VA supplemented diet, indicating an additive effect on mineralization. In conclusion, this study showed that the dietary increase in ARA and VA altered the skeletal metabolism during larval development, most likely through signaling pathways specific for each nutrient rather than an interaction. The present study also demonstrates that VA could affect the larval response to ARA, even within the accepted non-toxic/non-deficient range.

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.

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.

Toxic effects of dietary hydrolysed lipids: an in vivo study on fish larvae.

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.

Evidence for a regulatory loop between cholecystokinin (CCK) and tryptic enzyme activity in Atlantic cod larvae (Gadus morhua).

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.

The role of cholecystokinin and peptide YY in feed intake in Atlantic halibut (Hippoglossus hippoglossus) larvae.

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

Differential developmental toxicity of crude oil in early life stages of Atlantic halibut (Hippoglossus hippoglossus).

To further understand the complexity of developmental toxicity of dispersed oil and importance of exposure timing on fish early life stages, Atlantic halibut (Hippoglossus hippoglossus) were exposed to environmentally relevant concentrations through two embryonic developmental windows: the first period occurred during the epiboly process (named as "early embryonic exposure") and the second period overlapped the ontogenesis and cardiogenesis processes (named as "late embryonic exposure"). Following 72 hour oil exposure, embryos were transferred to clean seawater and a toxicity screening was performed in the yolk-sac larvae until first-feeding stages (56 days). The current study demonstrated that the exposure timing is essential for the development of toxic effects of crude oil in Atlantic halibut. Neither embryonic exposures (early or late) showed notable acute toxicity during exposure, yet both showed global latent teratogenic effects during yolk sac stages. Fish exposed during organogenesis (late) displayed stronger and more severe toxic effects than fish exposed during epiboly process (early), including reduced condition, severe craniofacial deformities and cardiovascular disruptions. The uptake level of polycyclic aromatic hydrocarbons into larval tissue and metabolic activity were greater following the late embryonic exposure and remained high during the depuration period at the highest exposure concentration. Overall, the long yolk sac stage development timing of Atlantic halibut makes this species a good candidate for evaluation of embryonic crude oil toxicity and its mechanisms.