Whole-genome re-sequencing provides key genomic insights in farmed Arctic charr (Salvelinus alpinus) populations of anadromous and landlocked origin from Scandinavia.

Arctic charr (Salvelinus alpinus) is a niche-market high-value species for Nordic aquaculture. Similar to other salmonids, both anadromous and landlocked populations are encountered. Whole-genome re-sequencing (22X coverage) was performed on two farmed populations of anadromous (Sigerfjord; n = 24) and landlocked (Arctic Superior; n = 24) origin from Norway and Sweden respectively. More than 5 million SNPs were used to study their genetic diversity and to scan for selection signatures. The two populations were clearly distinguished through principal component analysis, with the mean fixation index being ~0.12. Furthermore, the levels of genomic inbreeding estimated from runs of homozygosity were 6.23% and 8.66% for the Norwegian and the Swedish population respectively. Biological processes that could be linked to selection pressure associated primarily with the anadromous background and/or secondarily with domestication were suggested. Overall, our study provided insights regarding the genetic composition of two main strains of farmed Arctic charr from Scandinavia. At the same time, ample genomic resources were produced in the magnitude of millions of SNPs that could assist the transition of Nordic Arctic charr farming in the genomics era.

Digestive tract morphology and enzyme activities of juvenile diploid and triploid Atlantic salmon (Salmo salar) fed fishmeal-based diets with or without fish protein hydrolysates.

Triploid, sterile Atlantic salmon (Salmo salar) could make a contribution to the development of the farming industry, but uncertainties about the performance and welfare of triploids have limited their adoption by farmers. In this study, we compared the ontogeny of digestive tract morphology and enzyme activities (pepsin, trypsin, chymotrypsin, alkaline phosphatase and aminopeptidase) of diploid and triploid Atlantic salmon. Fish were fed diets based on fishmeal (STD) or a mix of fishmeal and hydrolysed fish proteins (HFM) whilst being reared at low temperature from start-feeding to completion of the parr-smolt transformation. Fish weights for each ploidy and feed combination were used to calculate thermal growth coefficients (TGCs) that spanned this developmental period, and the data were used to examine possible relationships between enzyme activities and growth. At the end of the experiment, faeces were collected and analyzed to determine the apparent digestibility coefficients (ADCs) of the dietary amino acids (AAs). Digestive tract histo-morphology did not differ substantially between ploidies and generally reflected organ maturation and functionality. There were no consistent differences in proteolytic enzyme activities resulting from the inclusion of HFM in the diet, nor was there improved digestibility and AA bioavailability of the HFM feed in either diploid or triploid fish. The triploid salmon had lower ADCs than diploids for most essential and non-essential AAs in both diets (STD and HFM), but without there being any indication of lower intestinal protease activity in triploid fish. When trypsin-to-chymotrypsin activity and trypsin and alkaline phosphatase (ALP) ratios (T:C and T:ALP, respectively) were considered in combination with growth data (TGC) low T:C and T:ALP values coincided with times of reduced fish growth, and vice versa, suggesting that T:C and T:ALP may be used to predict recent growth history and possible growth potential.

Transcriptome sequencing and histology reveal dosage compensation in the liver of triploid pre-smolt Atlantic salmon.

Triploid Atlantic salmon (Salmo salar L.) is seen as one of the best solutions to solve key issues in the salmon farming industry, such as the impact of escapees on wild stocks and pre-harvest sexual maturation. However, the effects of triploidy on salmon smoltification are poorly understood at the molecular level, even though smoltification is a very sensitive period that has a major influence on survival rate and performance of farmed salmon. In this study, we have compared the liver transcriptomes of diploid and triploid Atlantic salmon at three ontogeny stages: fry, parr and smolt. In diploid fish, a total of 2,655 genes were differentially expressed between fry and parr, whereas 506 genes had significantly different transcript levels between parr and smolts. In triploids, 1,507 and 974 genes were differentially expressed between fry and parr, and between parr and smolts, respectively. Most of these genes were down-regulated and 34 genes were differentially expressed between ploidies at the same stage. In both ploidy groups, the top differentially expressed genes with ontogeny stage belonged to common functional categories that can be related to smoltification. Nucleotide and energy metabolism were significantly down-regulated in fry when compared to parr, while immune system processes were significantly down-regulated in parr when compared to smolts. The close resemblance of enriched biological processes and pathways between ploidy groups suggests that triploidy is regulated by genome dosage compensation in Atlantic salmon. Histological analysis revealed that areas of vacuolization (steatosis) were present only in fry and parr stages, in contrast to a compact cellular histology with glycogen granules after smoltification. There was no significant difference in vacuolization between ploidy groups at the fry stage but the liver of diploid parr had a 33.5% higher vacuolization area compared to their triploid counterparts. Taken together, our data provide novel insights into the changes that occur at the molecular and histological level in the liver of both diploid and triploid Atlantic salmon prior to and during smoltification.

Growth and development of skeletal anomalies in diploid and triploid Atlantic salmon (Salmo salar) fed phosphorus-rich diets with fish meal and hydrolyzed fish protein.

Diploid and triploid Atlantic salmon, Salmo salar were fed high-protein, phosphorus-rich diets (56-60% protein; ca 18g phosphorus kg-1 diet) whilst being reared at low temperature from start-feeding until parr-smolt transformation. Performances of salmon fed diets based on fish meal (STD) or a mix of fishmeal and hydrolysed fish proteins (HFM) as the major protein sources were compared in terms of mortality, diet digestibility, growth and skeletal deformities. Separate groups of diploids and triploids were reared in triplicate tanks (initially 3000 fish per tank; tank biomass ca. 620 g) from 0-2745 degree-days post-start feeding (ddPSF). Growth metrics (weight, length, condition factor) were recorded at ca. 4 week intervals, external signs of deformities to the operculum, jaws and spinal column were examined in parr sampled at 1390 ddPSF, and external signs of deformity and vertebral anomalies (by radiography) were examined in fish sampled at the end of the trial (2745 ddPSF). The triploid salmon generally had a lower mass per unit length, i.e. lower condition factor, throughout the trial, but this did not seem to reflect any consistent dietary or ploidy effects on either dietary digestibility or the growth of the fish. By the end of the trial fish in all treatment groups had achieved a weight of 50+ g, and had completed the parr-smolt transformation. The triploids had slightly, but significantly, fewer vertebrae (Triploids STD 58.74 ± 0.10; HFM 58.68 ± 0.05) than the diploids (Diploids STD 58.97 ± 0.14; HFM 58.89 ± 0.01), and the incidence of skeletal (vertebral) abnormalities was higher in triploids (Triploids STD 31 ± 0.90%; HFM 15 ± 1.44%) than in diploids (Diploids STD 4 ± 0.80%; HFM 4 ± 0.83%). The HFM diet gave a significant reduction in the numbers of triploid salmon with vertebral anomalies in comparison with the triploids fed the STD diet possibly as a result of differences in phosphorus bioavailability between the two diets. Overall, the incidence of skeletal deformities was lower than reported in previous studies (Diploids 20+%, Triploids 40+%), possibly as a result of the combination of rearing at low-temperature and phosphorus-rich diets being used in the present study.

Flesh quality and biochemistry of light-manipulated Atlantic cod (Gadus morhua) and the significance of collagen cross-links on fillet firmness and gaping.

The aim of our study was to investigate whether light-manipulation, causing accelerated or delayed maturation, could be used as a tool to improve fillet gaping score and texture of farmed cod (Gadus morhua L.) harvested during summer. Control and accelerated groups had completed spawning by the time of harvest, and expressed sexual dimorphism in various biometric and flesh quality parameters. Pyridinoline cross-links, a key biochemical parameter investigated, were positively correlated with texture (r(2) = 0.4), as also were cathepsin B&L and sex. Gaping was also impacted by pyridinoline cross-links, together with body mass and maturation. Interestingly, the level of gaping was significantly reduced with a pyridinoline cross-link concentration of >2500 pmols g(-1) dry mass. In conclusion, light-manipulation can be used as a tool to improve flesh texture of farmed cod with a low gaping score, but with compromise of increased dress out percentage for the accelerated group.

Expression of growth-related genes in muscle during fasting and refeeding of juvenile Atlantic halibut, Hippoglossus hippoglossus L.

The aim of this study was to establish a fasting-refeeding protocol to investigate the expression of growth-related genes during the transition between catabolic and anabolic states in Atlantic halibut (Hippoglossus hippoglossus L.). Juveniles of approximately 950 g were maintained at ambient temperature (5-8 degrees C) until the 1st of May, then fasted for two months and refed for two months at 7.7-8.0 degrees C under continuous low light. Fast epaxial myotomal muscle was sampled at -64 d (days), -38 d, 0 d (start of refeeding), 3 d, 7 d, 14 d, 30 d and 60 d. Average body mass was unchanged over the fasting period but increased by 24.4% following 60 d refeeding. qPCR was used to analyse the stability of expression of five potential reference genes (Eef2, Fau, 18SrRNA, Actb and Tubb2) with GeNorm and Normfinder. Expression of the growth-related genes, cathepsin B (ctsb), cathepsin D (ctsd), insulin-like growth factor-I and II (IGF-I and II) and insulin-like growth factor-I receptor 1a (IGF-IRa) was normalised using the geometric average of the two most stable housekeeping genes, Fau and 18SrRNA. IGF-I mRNA showed a transient 2.6-fold increase in abundance with refeeding at 7 d whilst transcripts for IGF-II and IGF-IRa were elevated during fasting and decreased 3.8-fold and 3-fold between the 0 d and 3 d samples respectively. Ctsb expression increased between -64 d and 0 d and then decreased approximately 10-fold by 14 d refeeding. In contrast, ctsd was relatively unaffected by the fasting-refeeding cycle, showing a modest (approximately 35%) transient decrease in expression between the 0 d and 30 d refeeding samples. It was concluded that the experimental protocol adopted and housekeeping genes identified were suitable for investigating the catabolic-anabolic transition in halibut skeletal muscle.

Activity of aspargate (cathepsin D), cysteine proteases (cathepsins B, B + L, and H), and matrix metallopeptidase (collagenase) and their influence on protein and water-holding capacity of muscle in commercially farmed atlantic halibut (Hippoglossus hippoglossus L.).

Atlantic halibut (Hippoglossus hippoglossus L.) were commercially farmed in Helgeland, Norway (May 2004-May 2005). The average weight (Mb) of fish increased over the 12 month production cycle by approximately 73% for females and approximately 50% for males, although during the winter months (November-early May) Mb was unchanged in females and declined by 18% in males because of sexual maturation and sperm release. Periods of zero or negative growth were associated with up to 5.7% (females) and 17.9% (males) decline in fast muscle protein content. The activities of cathepsins B, B + L, H, and D showed a reciprocal relationship and were highly correlated with the changes in protein content. Water-holding capacity was measured as the liquid loss increased from 3-5% in November to 11-13% in May. Two general additive models (GAMs) showed that cathepsin B + L, cathepsin D, and collagenase explained 73.1% of the total variance in protein content, while cathepsin H was the largest contributor to liquid loss, explaining approximately 48.8% of the total variance. The results indicate that to obtain the best flesh quality Atlantic halibut should be harvested in the fall or early winter when the liquid loss and cathepsin activities are low and less likely to cause problems during secondary processing and storage.

Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development.

Gene expression studies are fundamental to understand the molecular basis of severe malformations in fish development, particularly under aquaculture conditions. Real-time PCR (qPCR) is the most accurate method of quantifying gene expression, provided that suitable endogenous controls are used to normalize the data. To date, no reference genes have been validated for developmental gene expression studies in Atlantic halibut (Hippoglossus hippoglossus). We have determined the expression profiles of 6 candidate reference genes (Actb, Eef2, Fau, Gapdh, Tubb2 and 18S rRNA) in 6 embryonic and 5 larval stages of Atlantic halibut development. There were significant changes in expression levels throughout development, which stress the importance and complexity of finding appropriate reference genes. The three software applications (BestKeeper, geNorm and NormFinder) used to evaluate the stability of potential reference genes produced comparable results. Tubb2 and Actb were the most stable genes across the different developmental stages, whereas 18S rRNA and Gapdh were the most variable genes and thus inappropriate to use as reference genes. According to geNorm and NormFinder, the best two-gene normalization factors corresponded to the geometric average of Tubb2/Actb and Tbb2/Fau, respectively. We believe that either of these normalization factors can be used for future developmental gene expression studies in Atlantic halibut.

Biochemical and structural factors contributing to seasonal variation in the texture of farmed Atlantic halibut (Hippoglossus hippoglossus L.) flesh.

Factors contributing to the texture of fish flesh, including pH, water content, density of fast muscle fibers, and the concentration of collagen and hydroxylysyl pyridinoline (PYD) cross-links, were investigated post-rigor in commercially farmed Atlantic halibut (Hippoglossus hippoglossus L.). The fish was sampled every quarter for a 12 month period from May 2004 to May 2005. Hydroxyproline (HYP) as a measure of collagen and PYD were determined using a high-performance liquid chromatography (HPLC) method. An ANCOVA model with fork length and season as covariates were used to explore the seasonal effects on texture, pH, muscle fiber density, alkaline-insoluble collagen (a-i HYP), alkaline-soluble collagen (a-s HYP), and PYD cross-links. A multiple linear regression (MLR) showed that the most important factors contributing to texture were PYD>water (%)>a-i HYP>fiber density, while pH and a-s HYP did not show any correlation to texture. The contribution of fast muscle fiber density to texture was found to vary between sexes and with the season, contributing more in males and in the spring. The most important parameter affecting texture was PYD, explaining 64% (p<0.001) of the total variation in a linear regression analysis. It is concluded that cross-linking processes are of great importance for the rigidity and strength of the collagen in Atlantic halibut flesh. Farmed halibut should be harvested in the fall or early winter when texture and nutrition are good to obtain optimal quality.