Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus

Background During the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. In this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. Despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene (HKG), depending on the tissue and experimental treatment, the gene has been us Results To establish a reliable method for the measurement of immune gene expression in Gasterosteus aculeatus, sequences from the now available genome database and an EST library of the same species were used to select oligonucleotide primers for HKG, in order to perform quantitative reverse-transcription (RT) PCR. The expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms BestKeeper, geNorm and N Conclusion As they were the most stably expressed genes in all tissues examined, we suggest using the genes for the L13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in Gasterosteus aculeatus.

Pharmacokinetics and transcriptional effects of the anti-salmon lice drug emamectin benzoate in Atlantic salmon (Salmo salar L.)

Background Emamectin benzoate (EB) is a dominating pharmaceutical drug used for the treatment and control of infections by sea lice (Lepeophtheirus salmonis) on Atlantic salmon (Salmo salar L). Fish with an initial mean weight of 132 g were experimentally medicated by a standard seven-day EB treatment, and the concentrations of drug in liver, muscle and skin were examined. To investigate how EB affects Atlantic salmon transcription in liver, tissues were assessed by microarray and qPCR at 7, 14 and 35 days after the initiation of medication. Results The pharmacokinetic examination revealed highest EB concentrations in all three tissues at day 14, seven days after the end of the medication period. Only modest effects were seen on the transcriptional levels in liver, with small fold-change alterations in transcription throughout the experimental period. Gene set enrichment analysis (GSEA) indicated that EB treatment induced oxidative stress at day 7 and inflammation at day 14. The qPCR examinations showed that medication by EB significantly increased the transcription of both HSP70 and glutathione-S-transferase (GST) in liver during a period of 35 days, compared to un-treated fish, possibly via activation of enzymes involved in phase II conjugation of metabolism in the liver. Conclusion This study has shown that a standard seven-day EB treatment has only a modest effect on the transcription of genes in liver of Atlantic salmon. Based on GSEA, the medication seems to have produced a temporary oxidative stress response that might have affected protein stability and folding, followed by a secondary inflammatory response.publishedVersio

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

An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV

<p>Abstract</p> <p>Background</p> <p>Due to the limited number of species specific antibodies against fish proteins, differential gene expression analyses are vital for the study of host immune responses. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most powerful tools for this purpose. Nevertheless, the accuracy of the method will depend on the careful selection of genes whose expression are stable and can be used as internal controls for a particular experimental setting.</p> <p>Findings</p> <p>The expression stability of five commonly used housekeeping genes [beta-actin (<it>ACTB</it>), elongation factor 1-alpha (<it>EF1A</it>), ubiquitin (<it>UBQ</it>), glyceraldehyd-3-phosphate dehydrogenase (<it>GAPDH</it>) and tubulin alpha (<it>TUBA</it>)] were monitored in salmonid cell lines CHSE-214 and RTS11 after infection with two of the most fastidious fish pathogens, the facultative bacterium <it>Piscirickettsia salmonis </it>and the aquabirnavirus IPNV (Infectious Pancreatic Necrosis Virus). After geNorm analysis, <it>UBQ </it>and <it>EF1A </it>appeared as the most stable, although <it>EF1A </it>was slightly upregulated at late stages of <it>P. salmonis </it>infection in RTS11. <it>ACTB </it>instead, showed a good performance in each case, being always considered within the three most stable genes of the panel. In contrast, infection-dependent differential regulation of <it>GAPDH </it>and <it>TUBA </it>was also demonstrated.</p> <p>Conclusion</p> <p>Based on the data presented here with the cell culture models CHSE-214 and RTS11, we suggest the initial choice of <it>UBQ</it>, <it>ACTB </it>and <it>EF1A </it>as reference genes in qRT-PCR assays for studying the effect of <it>P. salmonis </it>and IPNV on the host immune response.</p

Heart and Skeletal Muscle Inflammation of Farmed Salmon Is Associated with Infection with a Novel Reovirus

Atlantic salmon (Salmo salar L.) mariculture has been associated with epidemics of infectious diseases that threaten not only local production, but also wild fish coming into close proximity to marine pens and fish escaping from them. Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999[1], HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom[2]. Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful. Here we provide evidence that HSMI is associated with infection with piscine reovirus (PRV). PRV is a novel reovirus identified by unbiased high throughput DNA sequencing and a bioinformatics program focused on nucleotide frequency as well as sequence alignment and motif analyses. Formal implication of PRV in HSMI will require isolation in cell culture and fulfillment of Koch's postulates, or prevention or modification of disease through use of specific drugs or vaccines. Nonetheless, as our data indicate that a causal relationship is plausible, measures must be taken to control PRV not only because it threatens domestic salmon production but also due to the potential for transmission to wild salmon populations

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

No influence of oxygen levels on pathogenesis and virus shedding in Salmonid alphavirus (SAV)-challenged Atlantic salmon (Salmo salar L.)

<p>Abstract</p> <p>Background</p> <p>For more than three decades, diseases caused by salmonid alphaviruses (SAV) have become a major problem of increasing economic importance in the European fish-farming industry. However, experimental infection trials with SAV result in low or no mortality i.e very different from most field outbreaks of pancreas disease (PD). This probably reflects the difficulties in reproducing complex biotic and abiotic field conditions in the laboratory. In this study we looked at the relationship between SAV-infection in salmon and sub-lethal environmental hypoxia as a result of reduced flow-through in tank systems.</p> <p>Results</p> <p>The experiment demonstrated that constant reduced oxygen levels (60-65% oxygen saturation: 6.5-7.0 mg/L) did not significantly increase the severity or the progress of pancreas disease (PD). These conclusions are based upon assessments of a semi-quantitative histopathological lesion score system, morbidities/mortalities, and levels of SAV RNA in tissues and water (measured by 1 MDS electropositive virus filters and downstream real-time RT-PCR). Furthermore, we demonstrate that the fish population shed detectable levels of the virus into the surrounding water during viraemia; 4-13 days after i.p. infection, and prior to appearance of severe lesions in heart (21-35 dpi). After this period, viral RNA from SAV could not be detected in water samples although still present in tissues (gills and hearts) at lasting low levels. Lesions could be seen in exocrine pancreas at 7-21 days post infection, but no muscle lesions were seen.</p> <p>Conclusions</p> <p>In our study, experimentally induced hypoxia failed to explain the discrepancy between the severities reported from field outbreaks of SAV-disease and experimental infections. Reduction of oxygen levels to constant suboptimal levels had no effect on the severity of lesions caused by SAV-infection or the progress of the disease. Furthermore, we present a modified VIRADEL method which can be used to detect virus in water and to supplement experimental infection trials with information related to viral shedding. By using this method, we were able to demonstrate for the first time that shedding of SAV from the fish population into the surrounding water coincides with viraemia.</p

Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time reverse transcriptase PCR (RT-qPCR) has been widely used for quantification of mRNA as a way to determine key genes involved in different biological processes. For accurate gene quantification analysis, normalization of RT-qPCR data is absolutely essential. To date, normalization is most frequently achieved by the use of internal controls, often referred to as reference genes. However, several studies have shown that the reference genes used for the quantification of mRNA expression can be affected by the experimental set-up or cell type resulting in variation of the expression level of these key genes. Therefore, the evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of insects. For this purpose, ten candidate reference genes were investigated in three different tissues (midgut, Malpighian tubules, and fat body) of the oriental fruit fly, <it>Bactrocera dorsalis </it>(Hendel).</p> <p>Results</p> <p>Two different programs, <it>geNorm </it>and <it>Normfinder</it>, were used to analyze the data. According to <it>geNorm</it>, α-TUB + ACT5 are the most appropriate reference genes for gene expression profiling across the three different tissues in the female flies, while ACT3 + α-TUB are considered as the best for males. Furthermore, we evaluated the stability of the candidate reference genes to determine the sexual differences in the same tissue. In the midgut and Malpighian tubules, ACT2 + α-TUB are the best choice for both males and females. However, α-TUB + ACT1 are the best pair for fat body. Meanwhile, the results calculated by <it>Normfinder </it>are quite the same as the results with <it>geNorm</it>; α-TUB is always one of the most stable genes in each sample validated by the two programs.</p> <p>Conclusions</p> <p>In this study, we validated the suitable reference genes for gene expression profiling in different tissues of <it>B. dorsalis. </it>Moreover, appropriate reference genes were selected out for gene expression profiling of the same tissues taking the sexual differences into consideration. This work not only formed a solid basis for future gene expression study in <it>B. dorsalis</it>, but also will serve as a resource to screen reference genes for gene expression studies in any other insects.</p

Gene expression analyses of immune responses in Atlantic salmon during early stages of infection by salmon louse (Lepeophtheirus salmonis) revealed bi-phasic responses coinciding with the copepod-chalimus transition

The salmon louse (Lepeophtheirus salmonis Krøyer), an ectoparasitic copepod with a complex life cycle causes significant losses in salmon aquaculture. Pesticide treatments against the parasite raise environmental concerns and their efficacy is gradually decreasing. Improvement of fish resistance to lice, through biological control methods, needs better understanding of the protective mechanisms. We used a 21 k oligonucleotide microarray and RT-qPCR to examine the time-course of immune gene expression changes in salmon skin, spleen, and head kidney during the first 15 days after challenge, which encompassed the copepod and chalimus stages of lice development. Results Large scale and highly complex transcriptome responses were found already one day after infection (dpi). Many genes showed bi-phasic expression profiles with abrupt changes between 5 and 10 dpi (the copepod-chalimus transitions); the greatest fluctuations (up- and down-regulation) were seen in a large group of secretory splenic proteases with unknown roles. Rapid sensing was witnessed with induction of genes involved in innate immunity including lectins and enzymes of eicosanoid metabolism in skin and acute phase proteins in spleen. Transient (1-5 dpi) increase of T-cell receptor alpha, CD4-1, and possible regulators of lymphocyte differentiation suggested recruitment of T-cells of unidentified lineage to the skin. After 5 dpi the magnitude of transcriptomic responses decreased markedly in skin. Up-regulation of matrix metalloproteinases in all studied organs suggested establishment of a chronic inflammatory status. Up-regulation of putative lymphocyte G0/G1 switch proteins in spleen at 5 dpi, immunoglobulins at 15 dpi; and increase of IgM and IgT transcripts in skin indicated an onset of adaptive humoral immune responses, whereas MHCI appeared to be down-regulated. Conclusions Atlantic salmon develops rapid local and systemic reactions to L. salmonis, which, however, do not result in substantial level of protection. The dramatic changes observed after 5 dpi can be associated with metamorphosis of copepod, immune modulation by the parasite, or transition from innate to adaptive immune responses