Intestinal cellular localization of PCNA protein and CYP1A mRNA in Atlantic salmon Salmo salar L. exposed to a model toxicant

<p>Abstract</p> <p>Background</p> <p>The aim of the study was to examine the intestinal cellular localization of proliferating cell nuclear antigen (PCNA) and cytochrome P450 A1 (CYP1A) expression in Atlantic salmon <it>Salmo salar </it>L. exposed to a model toxicant. The stress response was induced by intraperitoneal injection of four salmon with a single dose (50 mg/kg) of the CYP1A inducer β-naphthoflavone (BNF) and intestinal tissue (mid and distal intestine; MI and DI) was sampled seven days later. Samples for histology and gene transcription analysis were collected from four exposed fish and four control fish. PCNA was assessed by immunohistochemistry, CYP1A mRNA was studied by <it>in situ </it>hybridization (ISH) and finally the transcription of five genes was quantified by real-time quantitative RT-PCR (real-time RT-PCR); two detoxifying genes (CYP1A and glutathione S-transferase; GST), a stress marker gene (heat shock protein 70; HSP70), PCNA and a gene marker of apoptosis (caspase 6A).</p> <p>Results</p> <p>PCNA protein and CYP1A mRNA were successfully localized in the intestinal cells (MI) of both experimental groups. At the cellular level, BNF significantly lowered intestinal cell proliferation and increased the CYP1A mRNA levels compared to the control group. The real-time RT-PCR data, which showed an increased mRNA expression both in the MI and DI of 139- and 62-fold, respectively, confirmed the increased cellular CYP1A mRNA levels detected using ISH. HSP70 expression was also up-regulated in the exposed fish. The other examined genes did not show any differential regulation in the experimental fish group.</p> <p>Conclusion</p> <p>This study showed that CYP1A mRNA had a specific intestinal cellular transcription pattern in Atlantic salmon exposed to BNF. At the cellular level CYP1A mRNA expression was always observed at or around the cell nucleus close to the basolateral cell membrane and at the tissue level CYP1A mRNA expression was most frequently observed in the basal and apex area of the intestinal folds. Taken together, a link between the intestinal detoxification system (CYP1A) and cell renewal system (PCNA) is indicated with these two processes being inversely correlated in BNF exposed fish.</p

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.

Characterization of an Atlantic cod (Gadus morhua) embryonic stem cell cDNA library

<p>Abstract</p> <p>Background</p> <p>The Atlantic cod is an ecologically and economically important North Atlantic fish species and also an emerging aquaculture species. To study gene expression in Atlantic cod embryonic stem (ES) cells, our goal was to generate and analyze expressed sequence tags (ESTs) from an ES cell cDNA library of mRNA consisting of approximately 3,900 ESTs.</p> <p>Results</p> <p>We sequenced 3,935 EST clones using a directional cDNA library made from pooled ES cells harvested at the blastula stage. Quality filtering of these ESTs allowed identification of 2,719 high-quality sequences with an average length of 442 bp containing 368 contigs and 1,276 singletons (1,644 unique sequences). BLASTX searches produced 889 significant (E-value < 10^-3) hits, of which 698 (42.5%) were annotated with Gene Ontology terms (E-value < 10^-6). The number of unknown unique sequences was 946 (57.5%). All the high-quality EST sequences have been deposited in GenBank (GenBank: 2,719 sequences in UniGene library dbEST id: 22,021). Gene discovery and annotations are presented and discussed.</p> <p>Conclusion</p> <p>This set of ESTs represents one of the first attempts to describe mRNA in ES cells from a marine cold-water fish species, and provides a basis for gene expression studies of Atlantic cod ES cells.</p

Selection of reference genes for qRT-PCR examination of wild populations of Atlantic cod Gadus morhua

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Do anesthetics and sampling strategies affect transcription analysis of fish tissues?

<p>Abstract</p> <p>Background</p> <p>The aim of the current examination was to evaluate if sedation and anesthetic treatment techniques affect the quality of RNA extracted from liver, gill, head kidney and brain tissues in Atlantic salmon <it>Salmo salar </it>L. Blood parameters were measured and tissue specimens sampled in six groups of fish; one control group (0 minutes), two groups kept in pure seawater in 90 liter tanks for 30 and 120 minutes, two groups treated with the anesthetic isoeugenol for 30 and 120 minutes, and one group kept in pure seawater for 105 minutes and then anaesthetized with metacaine for 15 minutes. RNA quality was assessed with the NanoDrop ND-1000 spectrophotometer (260/280 and 260/230 nm ratios) and with the Agilent Bioanalyzer (28S/18S ratio and RIN data) in samples either preserved in liquefied nitrogen (N₂) or in RNA<it>later</it>. In addition, the transcriptional levels of two fast-responding genes were quantified in gill and brain tissues.</p> <p>Results</p> <p>The results show that physiological stress during sampling does not affect the quality of RNA extracted from fish specimens. However, prolonged sedation (2 hours) resulted in a metabolic alkalosis that again affected the transcriptional levels of genes involved in ionoregulation and respiration. In gills, <it>Na</it>⁺-<it>K</it>⁺-<it>ATPase α1b </it>was significantly downregulated and <it>hypoxia inducible factor 1 </it>(<it>HIF1</it>) significantly upregulated after two hours of treatment with isoeugenol, suggesting that this commonly used sedative affects osmo-regulation and respiration in the fish. The results also suggest that for tissue preservation in general it is better to flash-freeze fish specimens in liquefied N₂than to use RNA<it>later</it>.</p> <p>Conclusion</p> <p>Prolonged sedation may affect the transcription of fast-responding genes in tissues of fish. Two hours of sedation with isoeugenol resulted in downregulation of the <it>Na</it>⁺-<it>K</it>⁺-<it>ATPase α1b </it>gene and upregulation of the <it>HIF1 </it>gene in gills of Atlantic salmon. The quality of RNA extracted from tissue specimens, however, was not affected by sedation treatment. Flash-freezing of tissue specimens seems to be the preferred preservation technique, when sampling fish tissue specimens for RNA extraction.</p

Spatial transcription of CYP1A in fish liver

<p>Abstract</p> <p>Background</p> <p>The aim of this work was to study how evenly detoxifying genes are transcribed spatially in liver tissue of fish. Ten Atlantic salmon <it>Salmo salar </it>were intraperitoneally injected with 50 mg/kg of the strong CYP1A inducer β-naphthoflavone and liver tissue harvested seven days later. The liver from 10 control and 10 exposed fish were split into eight sections, RNA extracted and three reference (β-actin, elongation factor 1A_B(EF1A_B)) and two detoxifying genes (CYP1A and GST) quantified with real-time RT-PCR. The cellular localization of the EF1A_Band CYP1A mRNA in the liver of control and β-naphthoflavone treated fish was then determined by <it>in situ </it>hybridization (ISH) using EF1A_Band CYP1A biotinylated oligonucleotide probes.</p> <p>Results</p> <p>The study shows that genes encoding phase I and phase II conjugating enzymes are unevenly transcribed in different parts of the liver of Atlantic salmon seven days after a single-dose of β-naphthoflavone exposure. Transcription of CYP1A and GST was higher in the middle section of the liver compared to the distal and proximal parts of the organ. The ISH data suggest that CYP1A transcription happens mainly in hepatocyte cells in the liver, and that hepatocytes in the vicinity of blood vessels respond stronger to β-naphthoflavone than cells further away from the blood supply.</p> <p>Conclusion</p> <p>Overall, the qRT-PCR and ISH results reported here suggest that gene expression analysis should be performed on as pure cell populations as possible. If bulk tissue samples are to be used, one should always check how evenly the target genes are expressed in tissue sections and organs in every study.</p

Lufenuron treatment temporarily represses gene expression and affects the SUMO pathway in liver of Atlantic salmon

Lufenuron is a benzoylurea insecticide currently in use to combat sea lice infestation in salmon aquaculture in Chile. With pending approval in Norway, the aim of this work was to study the uptake and toxicity of lufenuron in liver tissue of Atlantic salmon. Juvenile salmon weighing 40 g were given a standard 7-day oral dose, and bioaccumulation and transcriptional responses in the liver were examined 1 day after the end-of-treatment (day 8) and after 1 week of elimination (day 14). Bioaccumulation levels of lufenuron were 29 ± 3 mg/kg at day 8 and 14 ± 1 mg/kg at day 14, indicating relatively rapid clearance. However, residues of lufenuron were still present in the liver after 513 days of depuration. The exposure gave a transient inhibition of transcription in the liver at day 8 (2437 significant DEGs, p-adj < .05), followed by a weaker compensatory response at day 14 (169 significant DEGs). Pathways associated with RNA metabolism such as the sumoylation pathway were most strongly affected at day 8, while the apelin pathway was most profoundly affected at day 14. In conclusion, this study shows that lufenuron easily bioaccumulates and that a standard 7-day oral dose induces a transient inhibition of transcription in liver of salmon.publishedVersio

Short-term starvation at low temperature prior to harvest does not impact the health and acute stress response of adult Atlantic salmon

Period of starvation is regarded as a sound practice in aquaculture prior to handling, transportation and harvest, to minimise impacts on welfare and ensure proper hygiene after harvest. However, documentation of welfare issues such as stress following starvation and handling in adult Atlantic salmon are lacking. This study aimed to examine gut emptying and potential stress during a two weeks starvation period, and whether this starvation period changes the tolerance for physical stress. The study confirmed slower emptying of the gut segments at low temperature. Plasma and bile cortisol, and selected clinical analyses were used to characterize potential stress, as well as the response to acute physical crowding stress during the starvation period. Neither the general stress level nor the ability to cope with handling stress was affected by a 14 days starvation period. Down-regulation of selected nutritional related gene markers in liver indicated classical starvation responses, with reduced metabolism and oxidative pressure, and sparing of nutrients. The response to acute handling stress was not affected by two weeks of starvation. There were minor effects of starvation on stress and health markers, as evaluated by plasma lysozyme activity and gene expression of selected inflammation marker proteins in heart and skin tissues.</jats:p

Ontogeny-Specific Skeletal Deformities in Atlantic Haddock Caused by Larval Oil Exposure

Bone deformities are one of the main effects of crude oil exposure in marine fish larvae. Craniofacial and jaw deformities, if severe enough, may restrict feeding and ultimately kill the developing larvae. This study aimed to examine the impact of dispersed crude oil on bone development in Atlantic haddock (Melanogrammus aeglefinus) larvae, a fish species spawning in areas approached for oil and gas exploration in the North Atlantic Ocean. Atlantic haddock larvae were exposed to low (60 μg oil/L), high (600 μg oil/L), or pulsed (0–600, average 60 μg oil/L over time) dispersed crude oil from 0 to 18 days post hatch (dph). Endpoints included survival and growth, bone integrity, and transcriptional parameters, which were assessed during (0–18 dph) and after exposure until the fish reached 8 months of age (243 dph). The results showed that the larvae in the high treatment group had reduction in growth at 2–19, 44, 134, and 243 dph. Craniofacial abnormalities were most severe at 8 and 19 dph. These deformities were not present at 44 dph, possibly because the larvae with deformed jaws failed to feed properly and died. Higher prevalence of spinal deformities was observed in haddocks that survived for 243 dph. Three genes encoding proteins critical for osteoblast function, sp7, postn, and col10a1, were downregulated in the high treatment group larvae. We discuss possible mechanisms of action in the developing larvae after oil exposure. In conclusion, this study shows that larval exposure to oil can potentially have long-term effects on growth and bone integrity in Atlantic haddock.publishedVersio