Validation of reference genes for quantitative RT-qPCR studies of gene expression in Atlantic cod (Gadus morhua l.) during temperature stress

<p>Abstract</p> <p>Background</p> <p>One important physiological response to environmental stress in animals is change in gene expression. To obtain reliable data from gene expression studies using RT-qPCR it is important to evaluate a set of possible reference genes as normalizers for expression. The expression of these candidate genes should be analyzed in the relevant tissues during normal and stressed situations. To find suitable reference genes it was crucial that the genes were stably expressed also during a situation of physiological stress. For poikilotermic animals like cod, changes in temperature are normal, but if the changes are faster than physiological compensation, the animals respond with typical stress responses. It has previously been shown that Atlantic cod show stress responses when elevation of water temperature is faster than 1 degree/day, for this reason we chose hyperthermia as stress agent for this experiment.</p> <p>Findings</p> <p>We here describe the expression of eight candidate reference genes from Atlantic cod (<it>Gadus morhua l</it>.) and their stability during thermal stress (temperature elevation of one degree C/day for 5 days). The genes investigated were: Eukaryotic elongation factor 1 alpha, <it>ef1a</it>; 18s ribosomal RNA; <it>18s</it>, Ubiquitin conjugate protein; <it>ubiq</it>, cytoskeletal beta-actin; <it>actb</it>, major histcompatibility complex I; MHC-I light chain, beta-2 -microglobulin; <it>b2m</it>, cytoskeletal alpha-tubulin; <it>tba1c</it>, acidic ribosomal phosphoprotein; <it>rplp1</it>, glucose-6-phosphate dehydrogenase; <it>g6pd</it>. Their expression were analyzed in 6 tissues (liver, head kidney, intestine, spleen, heart and gills) from cods exposed to elevated temperature and compared to a control group. Although there were variations between tissues with respect to reference gene stability, four transcripts were more consistent than the others: <it>ubiq</it>, <it>ef1a</it>, <it>18s </it>and <it>rplp1</it>. We therefore used these to analyze the expression of stress related genes (heat shock proteins) induced during hyperthermia. We found that both transcripts were significantly upregulated in several tissues in fish exposed to increased temperature.</p> <p>Conclusion</p> <p>This is the first study comparing reference genes for RT-qPCR analyses of expression during hyperthermia in Atlantic cod. <it>ef1a, 18s, rplp1 </it>and <it>ubiq </it>transcripts were found to be well suited as reference genes during these experimental conditions.</p

Atlantic cod (Gadus morhua) hemoglobin genes: multiplicity and polymorphism

Background: Hemoglobin (Hb) polymorphism, assessed by protein gel electrophoresis, has been used almost exclusively to characterize the genetic structure of Atlantic cod (Gadus morhua) populations and to establish correlations with phenotypic traits such as Hb oxygen binding capacity, temperature tolerance and growth characteristics. The genetic system used to explain the results of gel electrophoresis entails the presence of one polymorphic locus with two major alleles (HbI-1; HbI-2). However, vertebrates have more than one gene encoding Hbs and recent studies have reported that more than one Hb gene is present in Atlantic cod. These observations prompted us to re-evaluate the number of Hb genes expressed in Atlantic cod, and to perform an in depth search for polymorphisms that might produce relevant phenotypes for breeding programs. Results: Analysis of Expressed Sequence Tags (ESTs) led to the identification of nine distinct Hb transcripts; four corresponding to the α Hb gene family and five to the β Hb gene family. To gain insights about the Hb genes encoding these transcripts, genomic sequence data was generated from heterozygous (HbI-1/2) parents and fifteen progeny; five of each HbI type, i.e., HbI-1/1, HbI-1/2 and HbI-2/2. β Hb genes displayed more polymorphism than α Hb genes. Two major allele types (β1A and β1B) that differ by two linked non-synonymous substitutions (Met55Val and Lys62Ala) were found in the β1 Hb gene, and the distribution of these β1A and β1B alleles among individuals was congruent with that of the HbI-1 and HbI-2 alleles determined by protein gel electrophoresis. RT-PCR and Q-PCR analysis of the nine Hb genes indicates that all genes are expressed in adult fish, but their level of expression varies greatly; higher expression of almost all Hb genes was found in individuals displaying the HbI-2/2 electrophoretic type. Conclusion: This study indicates that more Hb genes are present and expressed in adult Atlantic cod than previously documented. Our finding that nine Hb genes are expressed simultaneously in adult fish suggests that Atlantic cod, similarly to fish such as rainbow trout, carp, and goldfish, might be able to respond to environmental challenges such as chronic hypoxia or long-term changes in temperature by altering the level of expression of these genes. In this context, the role of the non-conservative substitution Lys62Ala found in the β1 Hb gene, which appears to explain the occurrence of the HbI-1 and HbI-2 alleles described by gel electrophoresis, and which was found to be present in other fish such as eel, emerald rockcod, rainbow trout and moray, requires further investigation

Thyroid and pituitary gland development from hatching through metamorphosis of a teleost flatfish, the Atlantic halibut

Fish larval development, not least the spectacular process of flatfish metamorphosis, appears to be under complex endocrine control, many aspects of which are still not fully elucidated. In order to obtain data on the functional development of two major endocrine glands, the pituitary and the thyroid, during flatfish metamorphosis, histology, immunohistochemistry and in situ hybridization techniques were applied on larvae of the Atlantic halibut (Hippoglossus hippoglossus), a large, marine flatfish species, from hatching through metamorphosis. The material was obtained from a commercial hatchery. Larval age is defined as day-degrees (D =accumulated daily temperature from hatching). Sporadic thyroid follicles are first detected in larvae at 142 D (27 days post-hatch), prior to the completion of yolk sack absorption. Both the number and activity of the follicles increase markedly after yolk sack absorption and continue to do so during subsequent development. The larval triiodothyronine (T3) and thyroxine (T4) content increases, subsequent to yolk absorption, and coincides with the proliferation of thyroid follicles. A second increase of both T3 and T4 occurs around the start of metamorphosis and the T3 content further increases at the metamorphic climax. Overall, the T3 content is lower than T4. The pituitary gland can first be distinguished as a separate organ at the yolk sack stage. During subsequent development, the gland becomes more elongated and differentiates into neurohypophysis (NH), pars distalis (PD) and pars intermedia (PI). The first sporadic endocrine pituitary cells are observed at the yolk sack stage, somatotrophs (growth hormone producing cells) and somatolactotrophs (somatolactin producing cells) are first observed at 121 D (23 days post-hatch), and lactotrophs (prolactin producing cells) at 134 D (25 days post-hatch). Scarce thyrotrophs are evident after detection of the first thyroid follicles (142 D ), but coincident with a phase in which follicle number and activity increase (260 D ). The somatotrophs are clustered in the medium ventral region of the PD, lactotrophs in the anterior part of the PD and somatolactotrophs are scattered in the mid and posterior region of the pituitary. At around 600 D , coinciding with the start of metamorphosis, somatolactotrophs are restricted to the interdigitating tissue of the NH. During larval development, the pituitary endocrine cells become more numerous. The present data on thyroid development support the notion that thyroid hormones may play a significant role in Atlantic halibut metamorphosis. The time of appearance and the subsequent proliferation of pituitary somatotrophs, lactotrophs, somatolactotrophs and thyrotrophs indicate at which stages of larval development and metamorphosis these endocrine cells may start to play active regulatory roles.This work has been carried out within the projects ‘‘Endocrine Control as a Determinant of Larval Quality in Fish Aquaculture’’ (CT-96-1422) and ‘‘Arrested development: The Molecular and Endocrine Basis of Flatfish Metamorphosis’’ (Q5RS-2002-01192), with financial support from the Commission of the European Communities. However, it does not necessarily reflect the Commission’s views and in no way anticipates its future policy in this area. This project was further supported by the Swedish Council for Agricultural and Forestry Research and Pluriannual funding to CCMAR by the Portuguese Science and Technology Council

The polycyclic aromatic hydrocarbons benzo[a]pyrene and phenanthrene inhibit intestinal lipase activity in rainbow trout (Oncorhynchus mykiss)

Contains fulltext : 175483.pdf (Publisher’s version ) (Open Access

Involvement of growth hormone-insulin-like growth factor I

The role of growth hormone (GH) and insulinlike growth factor-I (IGF-I) in the tissue remodeling associated with the transition of a symmetrical larva to an asymmetrical juvenile during flatfish metamorphosis is unknown. In order to investigate the potential role of these hormones in the remodeling of cranial bone and soft tissue that accompanies eye migration during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus) larvae, tissuespecific gene expression was monitored by in situ hybridization for Atlantic halibut type I growth hormone receptor (hhGHR), type II hhGHR, and insulin-like growth factor-I receptor (hhIGF-IR). Polyclonal antibody generated against the extracellular domain of type I hhGHR was used for the immunohistochemical localization of type I GHR protein. Type I hhGHR, type II hhGHR, and hhIGF-IR mRNA were expressed in fibroblasts, frontal bone osteocytes, and dorsal chondrocytes at the onset of metamorphosis (stage 8),during metamorphic climax (stage 9), and in fully metamorphosed juveniles (stage 10). Type I GHR protein showed similar expression patterns to those of type I hhGHR mRNA, except in chondrocytes in which little GHR protein was detected. The localization of GHR and IGF-IR transcripts and GHR protein in cranial structures that undergo remodeling is intriguing and suggests that, in addition to thyroid hormones, the GH-IGF-I system is involved in morphological transformations during metamorphosis in Atlantic halibut.We thank Heiddis Smáradóttir, Arnar Jónsson, and Øystein Saele for larval sampling, and Nádia Silva for methodological assistance