Christel Solberg

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.

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.