Transcription profiling by array of Atlantic cod head kidney cells following exposure to viral, bacterial and polyclonal activator stress
ABSTRACT: Understanding pathogen recognition and mechanisms in Atlantic cod are of significant importance for both basic research on wild populations and health management in aquaculture. A microarray approach was utilized to search for effects of viral (poly I:C/ polyinosinic acid:polycytidylic acid), bacterial (LPS/lipopolysaccharide) and polyclonal activator (PHA-L/phytohaemoagglutinin) stress in Atlantic cod head kidney cells. LPS cell activation increased mRNA expression of chemokine/Interleukin 8 (CXCL8/IL-8); interleukin -1? (IL-1?); cyclooxygenase 2 (COX2); leukocyte derived chemotaxin 2 (LECT2); LOC100698154, encoding a protein with unknown function; carboxyl-esterase 2 (CES2) and environmental biomarker cytochrome P450 1A (CYP1A). Mitogen activated protein kinase p38 (p38MAPK) and cathepsin F (CTSF) were downregulated by LPS. The antiviral responses induced by double stranded RNA (Poly I:C) clearly increased transcription of Toll like receptor 3 (TLR3) and interferon stimulating gene 15 (ISG15). The PHA response seemed to be more non-specific. Special for the PHA induction were the increase in Major histocompatibility complex class I (MHCI). CC chemokine type 2 (CK2) mRNA expression was increased by PHA, LPS and poly I:C, while p38MAPK and LECT2 was downregulated by PHA. Oxidative stress related genes like catalase and glutaredoxin (GLRX2) and the anti-apoptotic gene Bcl-2 showed no transcriptional changes compared to control in any of the treatments. Especially Poly I:C, but also LPS, induced leukotriene B4 (LTB4) and leukotriene B5 (LTB5) synthesis, while small amounts of prostaglandine E2 (PGE2) seemed to be constitutively produced in untreated cells, a production that was slightly elevated when exposing cells for LPS. This study reveals distinct signatures of bacteria and virus transcriptional responses in cod head kidney cells. In addition, the novel finding that Cyp1a was upregulated during the antibacterial response indicates a connection between immunity and aryl hydrocarbon receptor (AhR) activation in Atlantic cod.
Project description:This experiment was conducted in order to evaluate the potential contribution of oil droplets to the toxicity of dispersed oil to fish larvae. Atlantic cod larvae were exposed to five concentrations of either dispersed oil (D1-D5) (containing oil droplets [medium size 10-14 µm based on volume] and water soluble fraction [WSF]) or the filtered dispersion containing only WSF of oil (W1-W5) for four days and harvested for transcriptional analysis at 13 days post hatching. The most significant differently expressed genes were observed in cod larvae exposed to the highest concentration of the dispersed oil (containing 10.41 ± 0.46 µg ∑PAH/L), with CYP1A showing the strongest response. Functional analysis further showed that the top scored network as analyzed with Ingenuity Pathway Analysis was “Drug Metabolism, Endocrine System Development and Function, Lipid Metabolism”. Oil exposure also increased the expression of genes involved in bone resorption and decreased the expression of genes related to bone formation. In conclusion, oil exposure affects drug metabolism, endocrine regulation, cell differentiation and proliferation, apoptosis, fatty acid biosynthesis and tissue development in Atlantic cod larvae. The altered gene transcription was dominated by the WSF and the oil droplet fraction only had a moderate impact on the observed changes.
Project description:Female Atlantic cod (Gadus morhua)were devided in to five groups and oraly exposed to alkylphenols and produced water for 20 weeks. Differentially expressed genes were studied in liver samples using the CodStress array. The five groups were fed with feed-paste containing two different doses of the AP mixture (Low AP and High AP), PW, 17?-estradiol (E2) and toxicant-free paste (control group) respectively. The body burden for each compound corresponded to 20 ?g/kg of total AP in Low AP group, 4000 ?g/kg ?g/kg of total AP in High AP group, 100 ?g E2/kg in the E2 group 500 mg PW/kg in the PW group.
Project description:The Atlantic cod, Gadus morhua, is an important species both for traditional fishery and increasingly also in fish farming. The Atlantic cod is also under potential threat from various environmental changes such as pollution and climate change, but the biological impact of such changes are not well known, in particular when it comes to sublethal effects that can be difficult to assert. Modern molecular and genomic approaches have revolutionized biological research during the last decade, and offer new avenues to study biological functions and e.g. the impact of anthropogenic activities at different life-stages for a given organism. In order to develop genomic data and genomic tools for Atlantic cod we conducted a program were we constructed 20 cDNA libraries, and produced and analyzed 44006 expressed sequence tags (ESTs) from these. Several tissues are represented in the multiple cDNA libraries, that differ in either sexual maturation or immulogical stimulation. This approach allowed us to identify genes that are expressed in particular tissues, life-stages or in response to specific stimuli, and also gives us information about potential functions of the transcripts. The ESTs were used to construct a 16 k cDNA microarray to further investigate the cod transcriptome. Microarray analyses were preformed on pylorus, pituitary gland, spleen and testis of sexually maturing male cod. The four different tissues displayed tissue specific transcriptomes demonstrating that the cDNA array is working as expected and will prove to be a powerful tool in further experiments.
Project description:In the present study we aimed to develop a custom made cDNA microarray, the CodStress array, for Atlantic cod to be used as a diagnostic tool in environmental monitoring of polluted waters. We wanted to investigate if the gene expression profiles would reflect environmental levels and composition of pollutants in two saltwater recipients, S?rfjorden and Store Lungeg?rdsvann.
Project description:This study was conduct to identify the virus-responsive transcripts in Atlantic cod, using viral mimic, polyriboinosinic polyribocytidylic acid (pIC) Overall design: Six individual Atlantic cod (1.087 ± 0.73 kg) were used for identification of antiviral (i.e. pIC-responsive) macrophage transcripts. The fish were kept in a 21 m^3 tank and optimum conditions (5.2-6.4˚C, 95-110% oxygen saturation and under an ambient photoperiod) in the Dr. Joe Brown Aquatic Research Building (JBARB) of the Ocean Sciences Centre (OSC). Atlantic cod head kidney cells were isolated from 6 fish. The cells were allowed to adhere overnight (16 h) at 10˚C, and then the non-adherent cells were removed. Thereafter, macrophages (adherent cells) from each individual were exposed to either viral mimic using 50 ug/ml pIC or PBS (control). Macrophages were lysed at 24 HPS using TRIzol, and RNAs were extracted. A common reference design was used for the microarray experiment. For each group (pIC or control), 6 biological replicates (pIC and control from 6 fish) were used for microarray experiment. The common reference consisted of a pool of 12 RNA samples. All test samples were labeled with Cy5, whereas common reference was labeled with Cy3. Each individual test sample was co-hybridized with the common reference sample on an array (20K Atlantic cod microarray platform); therefore, 12 arrays were used for this experiment. [variable] other: pIC: Fish_1_pIC, Fish_2_pIC, Fish_3_pIC, Fish_4_pIC, Fish_5_pIC, Fish_6_pIC, [variable] other: Control: Fish_1_Control, Fish_2_Control, Fish_3_Control, Fish_4_Control, Fish_5_Control, Fish_6_Control, [repeat] biological replicate: Fish_1_pIC, Fish_2_pIC, Fish_3_pIC, Fish_4_pIC, Fish_5_pIC, Fish_6_pIC, [repeat] biological replicate: Fish_1_Control, Fish_2_Control, Fish_3_Control, Fish_4_Control, Fish_5_Control, Fish_6_Control,
Project description:This study was performed to validate the newly developed CGP Atlantic cod 20K oligonucleotide microarray. Atlantic cod (Gadus morhua) received an intraperitoneal injection of either formalin-killed, atypical Aeromonas salmonicida (Asal) or PBS and transcriptional profiles of spleen tissues from Asal-injected fish were compared to those from pre-injection control fish and PBS-injected control fish. Gene expression profiles resulting from this study were compared to those from suppression subtractive hybridization library studies, that were previously performed on the same samples, and to literature. Gene expression patterns of single genes were confirmed by QPCR analysis. This study has shown that the newly developed CGP Atlantic cod 20K oligo microarray platform is a valuable tool for cod genomic research. Overall design: Fish were assigned to 1 of 3 groups: Asal group, PBS group or 'undisturbed control' group (the latter were not handled during the experiment). These groups were kept in 3 separate tanks. For the test samples, RNA was used from individual spleen samples that were taken from 6 fish from each of 4 treatment groups: PBS group pre-injection (0H PBS), Asal group pre-injection (0H Asal), PBS group 24 hours post-injection (24HPI PBS) and Asal group 24 hours post-injection (24HPI Asal). All test samples were labeled with AlexaFluor 647. For the universal reference sample, RNA from 31 'undisturbed control' fish was pooled, with each individual contributing an equal amount, and labeled with AlexaFluor 555. Each individual test sample was hybridized together with the universal reference sample on an array. For one sample from the 0H PBS group the array failed. This study therefore includes 6 biological replicates for 0H Asal, 24HPI Asal and 24HPI PBS groups and 5 biological replicates for the 0H PBS group.