Thermal delousing has become the most applied method for treatment against salmon lice. However, the tem-perature range used is strongly aversive for salmonids, and the method is associated with increased mortality. Treatment temperature x duration combinations should be tailored to maximise delousing efficiency and minimize welfare impacts on the host fish. We tested the detachment rate of sessile, pre-adult and adult male and female salmon lice (Lepeophtheirus salmonis) as a function of ambient temperature (11-16 degrees C), exposure tem-perature (28-36 degrees C), and exposure duration (0-120 s). Dead Atlantic salmon (Salmo salar) hosts were used to avoid negative fish welfare and detachment of lice due to fish behaviour. Within the range tested, higher exposure temperatures were associated with higher detachment rates among pre-adult and adult lice, while no sessile lice detached at any temperature. Moreover, no treatment combination detached 100% of lice of any stage, and at 28 degrees C, detachment of adult females was negligible. Most detachments occurred within the first 30 s of exposure. We conclude that for a given delousing efficiency, lower temperatures must be compensated for by considerably longer exposure durations. This may be a higher risk for the welfare of the host fish than higher temperatures and shorter exposure durations.

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.

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.

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.

ABSTRACTBackgroundReproductive success and normal development in all animals are dependent on egg quality and developmental competence of the produced embryo. This study employed tandem mass tags labeling based liquid chromatography tandem mass spectrometry for egg proteomic profiling to investigate differences in the global proteome of good versus poor quality Atlantic halibut eggs at 1-cell stage post fertilization.ResultsA total of 115 proteins were found to be differentially abundant between good and poor quality eggs. Frequency distribution of these proteins revealed higher protein folding activity in good quality eggs in comparison to higher transcription and protein degradation activities in poor quality eggs (p< 0.05). Poor quality halibut eggs were significantly enriched with additional proteins related to mitochondrial structure and biogenesis (p< 0.05). The differential abundance of a selection of proteins was first confirmed at gene expression level using a transcriptomic approach followed by a targeted proteomic approach (parallel reaction monitoring based mass spectrometry) in biological samples obtained from two consecutive reproductive seasons. The findings of global proteome profiling, together with the validation of differential abundance of targeted proteins and their related genes, suggest impairments in protein and energy homeostasis which might be related to unfolded protein response and mitochondrial stress in poor quality eggs. Additional transmission electron microscopy studies were taken to assess potential differences in abundance and morphological integrity of mitochondria between good and poor quality eggs. Observations reveal poor quality eggs to contain significantly higher number of mitochondria with higher number of cristae. These mitochondria, however, are significantly smaller and have a more irregular shape than those found in high-quality eggs. Therewithal difference in mtDNA levels represented bymt-nd5andmt-atp6genomic DNA abundance in this study, were found to be not statistically significant (p> 0.05) between good and bad quality eggs at both 1 hpf and 24 hpf stages.ConclusionOverall evidence from this study indicate that poor quality eggs undergo impairments at both transcription and translation level leading to endoplasmic reticulum and mitochondrial deficiencies. Additional research may be required to expediate the details and the potential of these impairments occurring in different species. Nonetheless, this study will pave the way for future research and will help in acceleration of recent advances in the field of embryonic developmental competence of living organisms.

The in vivo ability of Atlantic halibut (Hippoglossus hippoglossus) larvae to incorporate, de-acylate and re-acylate [1-14C]fatty acids (FA) into lipid classes and to elongate and desaturate those substrates was elucidated. To this purpose, 30 days post-first-feeding (30 dpff) larvae were incubated in the presence of either free [1-14C]-FAs (18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3), [1-14C]18:1n-9 bound to mono- (MAG) and triacylglycerols (TAG), or [1-14C]20:4n-6 bound to phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Halibut larvae can efficiently incorporate dietary FAs through MAG, while FA incorporation when provided as free FAs and especially bound to TAG or phospholipids seems to be very low. These results suggest that lipid digestion might be a limiting factor in the FA absorption and incorporation by 30 dpff Atlantic halibut. The present study also shows that esterification into phospholipids is a strong metabolic fate for dietary long-chain polyunsaturated fatty acids (LC-PUFAs), and the capacity of 30 dpff Atlantic halibut larvae to metabolize dietary FAs through elongation/desaturation processes. Nonetheless, neither EPA nor DHA were detected from [1-14C]ALA, indicating a reduced capacity to biosynthesize these LC-PUFAs and the necessity for them to be adequately supplied through the diet for proper larval development.

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.

Major challenges in culture of Atlantic halibut larvae have been slow growth during the late larval stages and inferior juvenile quality due to pigmentation errors and incomplete eye migration during metamorphosis. The hypothesis of this study was that feeding on-grown Artemia would alleviate these problems. Artemia were grown for 3-4 days on Origreen or Origo. The growth and nutrient composition of Artemia nauplii and on-grown Artemia were analysed, and both Artemia types were fed to Atlantic halibut larvae, on-grown Artemia from 15 days post-first feeding (dpff). The body length of Artemia increased with 20%-70% in response to on-growing. In all experiments, protein, free amino acids and the ratio of phospholipid to total lipid increased, while lipid and glycogen decreased. The fatty acid composition improved in some cases and not in others. The micronutrient profiles were not negatively affected in on-grown Artemia. All these changes are thought to be beneficial for marine fish larvae. The final weight of Atlantic halibut postlarvae was similar, and 90% of the juveniles had complete eye migration in both groups. It is concluded that the present version of Artemia nauplii probably covers the nutrient requirements of Atlantic halibut larvae.

Atlantic halibut is a cold-water pleuronectiform of the North Atlantic with a high market price and a small wild stock, therefore it has a large potential in farming. However, the halibut is difficult to grow and although work aimed at domestication has been going on since the late 1980ies, there are still only a few commercial farms. The purpose of the present study was to establish a time-point for early weaning of Atlantic halibut larvae, using feed intake as a proxy. First, three commercial diets (Otohime, Gemma Micro and AgloNorse) were compared at 28 days post first-feeding (dpff), with respect to feed intake during 5 days after weaning, using four replicate tanks. The number of larvae per tank out of 28 which had filled guts on the different diets, was 26.7 +/- 2.3 (Otohime), 12.0 +/- 0.6 (Gemma Micro) and 14.7 +/- 1.2 (AgloNorse), respectively. Otohime was chosen for an experiment where feed intake for 5 days after weaning was monitored, starting at 15, 22 and 28 dpff. There was a gradual increase in success with larval age. At 15 dpff the larvae did not eat and there was an almost 100% mortality, at 22 dpff, 12 +/- 1 out of 17 larvae survived and 7.3 +/- 1.5 had filled guts. At 28 dpff, 14 +/- 3 out of 17 larvae survived and all of them had filled guts. The development seems to correlate with the development of larval type digestion. Based on feed intake, successful weaning can be achieved at 28 dpff, however, further studies are needed to determine growth, survival and juvenile quality of Atlantic halibut larvae that have been weaned early on formulated diets.

Species Leader: Dr. Birgitta Norberg (Institute of Marine Research, Norway), Other Scientists participating: Constantinos C. Mylonas (Hellenic Center for Marine Research, Greece), Torstein Harboe (Institute of Marine Research, Norway), Kristin Hamre (Institute of Marine Research, Norway), Ramon Fontanillas (Skretting ARC, Norway), Audun Nerland (Institute of Marine Research, Norway), Sonal Patel (Institute of Marine Research, Norway), Rocio Robles (Ctaqua).

Sea cage fish aquaculture attracts large aggregations of wild fish that feed on farm waste.Fish that associate closely with farms undergo physiological changes, and captive feeding trials indicate possible negative effects on reproductive fitness.However, little is known about the significance of this phenomenon for reproduction in wild fish over larger spatial scales.To assess whether coastal areas with intensive aquaculture impact the fitness of wild fish, we collected Atlantic cod Gadus morhua from 2 areas, 1 with high and 1 with low Atlantic salmon Salmo salar farming density (HFD and LFD, respectively) in southwestern Norway, a region that hosts the world's largest coastal fish aquaculture industry.We conducted a captive spawning trial and compared a range of reproductive fitness metrics.Two fatty acids that occur at high levels in commercial feed, oleic and lineoleic acid, were strongly correlated in the ovaries of HFD fish, but a comparison of lipid profiles between HFD and LFD fish showed no differences in total lipids or essential fatty acids.Although HFD fish were slightly larger than LFD fish and had similar body condition, the volume of eggs produced did not differ, indicating relatively lower reproductive investment by HFD fish.HFD eggs were 5% smaller than LFD eggs, which did not lead to differential hatching success but may have contributed to HFD larvae being 8% smaller than their LFD counterparts at 40 d post spawning.The potential for cumulative effects of smaller eggs and larvae on fitness at later life stages warrants further investigation.

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.

Deiodinase 3 (Dio3) plays an essential role during early development in vertebrates by controlling tissue thyroid hormone (TH) availability. The Atlantic halibut (Hippoglossus hippoglossus) possesses duplicate dio3 genes (dio3a and dio3b). Expression analysis indicates that dio3b levels change in abocular skin during metamorphosis and this suggests that this enzyme is associated with the divergent development of larval skin to the juvenile phenotype. In larvae exposed to MMI, a chemical that inhibits TH production, expression of dio3b in ocular skin is significantly up-regulated suggesting that THs normally modulate this genes expression during this developmental event. The molecular basis for divergent dio3a and dio3b expression and responsiveness to MMI treatment is explained by the multiple conserved TREs in the proximal promoter region of teleost dio3b and their absence from the promoter of dio3a. We propose that the divergent expression of dio3 in ocular and abocular skin during halibut metamorphosis contributes to the asymmetric pigment development in response to THs.

The increasing amount of dissolved anthropogenic CO2 has caused a drop in pH values in the open ocean known as ocean acidification. This change in seawater carbonate chemistry has been shown to have a negative effect on a number of marine organisms. Early life stages are the most vulnerable, and especially the organisms that produce calcified structures in the phylum Mollusca. Few studies have looked at effects on scallops, and this is the first study presented including fed larvae of the great scallop (Pecten maximus) followed until day 14 post-fertilization. Fertilized eggs from unexposed parents were exposed to three levels of pCO(2) using four replicate units: 465 (ambient), 768 and 1294 mu atm, corresponding to pH(NIST) of 7.94, 7.75 (-0.19 units) and 7.54 (-0.40 units), respectively. All of the observed parameters were negatively affected by elevated pCO(2): survival, larval development, shell growth and normal shell development. The latter was observed to be affected only 2 days after fertilization. Negative effects on the fed larvae at day 7 were similar to what was shown earlier for unfed P. maximus larvae. Growth rate in the group at 768 mu atm seemed to decline after day 7, indicating that the ability to overcome the environmental change at moderately elevated pCO(2) was lost over time. The present study shows that food availability does not decrease the sensitivity to elevated pCO(2) in P. maximus larvae. Unless genetic adaptation and acclimatization counteract the negative effects of long term elevated pCO(2), recruitment in populations of P. maximus will most likely be negatively affected by the projected drop of 0.06-0.32 units in pH within year 2100.

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 3h 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 30min after refeeding, while ghrelin, PYY and NPY transcript expression increased 2, 4 and 5h 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.

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.

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 β 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.

The ontogenetic development of the gut hormone cholecystokinin (CCK) and the key proteolytic enzyme trypsin was described in Atlantic cod larvae (Gadus morhua) from first-feeding until 38days post first-feeding (dpff). CCK is known to play a major role in the endocrine control of digestive processes in mammals and adult fish, but its regulatory role in the larval stages of marine fish is largely unknown. Only small amounts of CCK were found in the body (excluding head) in cod larvae at first-feeding, but CCK levels increased exponentially with development, suggesting a more pronounced role of CCK during ontogeny. Tryptic enzyme activity increased slightly until a standard length of ca. 8mm (approx. 33days dpff) with a significant increase in larvae larger than 8mm standard length, indicating limited digestive capacity in the early stages. To entangle the short-term feedback mechanism between CCK and tryptic enzyme activity, we conducted a 12hour feeding experiment at 21 dpff. Cod larvae receiving only algae revealed a noticeable response in tryptic enzyme activity within two hours in the morning, whereas larvae fed algae and rotifers at the same time showed a slightly delayed response up to four hours. Tryptic enzyme activity remained low in the group receiving only algae as well as the two fed groups in the afternoon. No reaction in tryptic enzyme activity was observed in larvae that received a second meal of rotifers in the afternoon, indicating limited regulatory and digestive capacity to handle several meals in a short period. CCK levels remained relatively constant throughout the day but increased in the afternoon in all three groups when tryptic enzyme activity was low, suggesting that a negative feedback mechanism between CCK and tryptic enzyme activity is present in larval cod at least from 21 dpff.

Introduction Conclusions References