Marije Booman

A moderate increase in ambient temperature modulates the Atlantic cod (Gadus morhua) spleen transcriptome response to intraperitoneal viral mimic injection

BackgroundAtlantic cod (Gadus morhua) reared in sea-cages can experience large variations in temperature, and these have been shown to affect their immune function. We used the new 20K Atlantic cod microarray to investigate how a water temperature change which, simulates that seen in Newfoundland during the spring-summer (i.e. from 10°C to 16°C, 1°C increase every 5 days) impacted the cod spleen transcriptome response to the intraperitoneal injection of a viral mimic (polyriboinosinic polyribocytidylic acid, pIC).ResultsThe temperature regime alone did not cause any significant increases in plasma cortisol levels and only minor changes in spleen gene transcription. However, it had a considerable impact on the fish spleen transcriptome response to pIC [290 and 339 significantly differentially expressed genes between 16°C and 10°C at 6 and 24 hours post-injection (HPI), respectively]. Seventeen microarray-identified transcripts were selected for QPCR validation based on immune-relevant functional annotations. Fifteen of these transcripts (i.e. 88%), including DHX58, STAT1, IRF7, ISG15, RSAD2 and IκBα, were shown by QPCR to be significantly induced by pIC.ConclusionsThe temperature increase appeared to accelerate the spleen immune transcriptome response to pIC. We found 41 and 999 genes differentially expressed between fish injected with PBS vs. pIC at 10°C and sampled at 6HPI and 24HPI, respectively. In contrast, there were 656 and 246 genes differentially expressed between fish injected with PBS vs. pIC at 16°C and sampled at 6HPI and 24HPI, respectively. Our results indicate that the modulation of mRNA expression of genes belonging to the NF-κB and type I interferon signal transduction pathways may play a role in controlling temperature-induced changes in the spleen’s transcript expression response to pIC. Moreover, interferon effector genes such as ISG15 and RSAD2 were differentially expressed between fish injected with pIC at 10°C vs. 16°C at 6HPI. These results substantially increase our understanding of the genes and molecular pathways involved in the negative impacts of elevated ambient temperature on fish health, and may also be valuable to our understanding of how accelerated global climate change could impact cold-water marine finfish species.

An Integrated Approach to Gene Discovery and Marker Development in Atlantic Cod (Gadus morhua)

Atlantic cod is a species that has been overexploited by the capture fishery. Programs to domesticate this species are underway in several countries, including Canada, to provide an alternative route for production. Selective breeding programs have been successfully applied in the domestication of other species, with genomics-based approaches used to augment conventional methods of animal production in recent years. Genomics tools, such as gene sequences and sets of variable markers, also have the potential to enhance and accelerate selective breeding programs in aquaculture, and to provide better monitoring tools to ensure that wild cod populations are well managed. We describe the generation of significant genomics resources for Atlantic cod through an integrated genomics/selective breeding approach. These include 158,877 expressed sequence tags (ESTs), a set of annotated putative transcripts and several thousand single nucleotide polymorphism markers that were developed from, and have been shown to be highly variable in, fish enrolled in two selective breeding programs. Our EST collection was generated from various tissues and life cycle stages. In some cases, tissues from which libraries were generated were isolated from fish exposed to stressors, including elevated temperature, or antigen stimulation (bacterial and viral) to enrich for transcripts that are involved in these response pathways. The genomics resources described here support the developing aquaculture industry, enabling the application of molecular markers within selective breeding programs. Marker sets should also find widespread application in fisheries management.

Development and Experimental Validation of a 20K Atlantic Cod (Gadus morhua) Oligonucleotide Microarray Based on a Collection of over 150,000 ESTs

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.

Atlantic salmon (Salmo salar) liver transcriptome response to diets containing Camelina sativa products.

Due to increasing demand for fish oil (FO) and fish meal (FM) in aquafeeds, more sustainable alternatives such as plant-derived oils and proteins are needed. Camelina sativa products are viable feed ingredients given the high oil and crude protein content in the seed. Atlantic salmon were fed diets with complete or partial replacement of FO and/or FM with camelina oil (CO) and/or camelina meal (CM) in a 16-week trial [Control diet: FO; Test diets: 100% CO replacement of FO (100CO), or 100CO with solvent-extracted FM (100COSEFM), 10% CM (100CO10CM), or SEFM+10% CM (100COSEFM10CM)]. Diet composition, growth, and fatty acid analyses for this feeding trial were published previously. A 44K microarray experiment identified liver transcripts that responded to 100COSEFM10CM (associated with reduced growth) compared to controls, yielding 67 differentially expressed features (FDR<5%). Ten microarray-identified genes [cpt1, pcb, bar, igfbp-5b (2 paralogues), btg1, dnph1, lect-2, clra, klf9, and fadsd6a], and three additional genes involved in lipid metabolism [elovl2, elovl5 (2 paralogues), and fadsd5], were subjected to QPCR with liver templates from all 5 dietary treatments. Of the microarray-identified genes, only bar was not QPCR validated. Both igfbp-5b paralogues were significantly down-regulated, and fadsd6a was significantly up-regulated, in all 4 camelina-containing diet groups compared with controls. Multivariate statistics were used to correlate hepatic desaturase and elongase gene expression data with tissue fatty acid profiles, indicating the involvement of these genes in LC-PUFA biosynthesis. This nutrigenomic study provides molecular biomarkers for use in developing novel aquafeeds using camelina products.