Project description:Background: Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all animals are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K+–ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. Results: No differences in Gill Na+, K+–ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. Conclusion: While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards either an unresolved infection or a nutritional deficiency as underlying drivers of this phenotype.

Project description:The sea-run phenotype of rainbow trout (Oncorhynchus mykiss), like other anadromous salmonids, present a juvenile stage fully adapted to life in freshwater known as parr. Development in freshwater is followed by the smolt stage, where preadaptations needed for seawater life are developed making fish ready to migrate to the ocean, after which event they become post-smolts. While these three life stages have been studied using a variety of approaches, proteomics has never been used for such purpose. The present study characterised the blood plasma proteome of parr, smolt and post-smolt rainbow trout using a gel electrophoresis liquid chromatography tandem mass spectrometry approach alone or in combination with low-abundant protein enrichment technology (combinatorial peptide ligand library). In total, 1,822 proteins were quantified, 17.95% of them being detected only in plasma post enrichment. Across all life stages, the most abundant proteins were ankyrin-2, DNA primase large subunit, actin, serum albumin, apolipoproteins, hemoglobin subunits, hemopexin-like proteins and complement C3. When comparing the different life stages, 17 proteins involved in mechanisms to cope with hyperosmotic stress and retinal changes, as well as the downregulation of nonessential processes in smolts, were significantly different between parr and smolt samples. On the other hand, 11 proteins related to increased growth in post-smolts, and also related to coping with hyperosmotic stress and to retinal changes, were significantly different between smolt and post-smolt samples. Overall, this study presents a series of proteins with the potential to complement current seawater-readiness assessment tests in rainbow trout, which can be measured non-lethally in an easily accessible biofluid. Furthermore, this study represents a first in-depth characterisation of the rainbow trout blood plasma proteome, having considered three life stages of the fish and used both fractionation alone or in combination with enrichment methods to increase protein detection.

Project description:BackgroundGenetic maps characterizing the inheritance patterns of traits and markers have been developed for a wide range of species and used to study questions in biomedicine, agriculture, ecology and evolutionary biology. The status of rainbow trout genetic maps has progressed significantly over the last decade due to interest in this species in aquaculture and sport fisheries, and as a model research organism for studies related to carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. We constructed a second generation genetic map for rainbow trout using microsatellite markers to facilitate the identification of quantitative trait loci for traits affecting aquaculture production efficiency and the extraction of comparative information from the genome sequences of model fish species.ResultsA genetic map ordering 1124 microsatellite loci spanning a sex-averaged distance of 2927.10 cM (Kosambi) and having 2.6 cM resolution was constructed by genotyping 10 parents and 150 offspring from the National Center for Cool and Cold Water Aquaculture (NCCCWA) reference family mapping panel. Microsatellite markers, representing pairs of loci resulting from an evolutionarily recent whole genome duplication event, identified 180 duplicated regions within the rainbow trout genome. Microsatellites associated with genes through expressed sequence tags or bacterial artificial chromosomes produced comparative assignments with tetraodon, zebrafish, fugu, and medaka resulting in assignments of homology for 199 loci.ConclusionThe second generation NCCCWA genetic map provides an increased microsatellite marker density and quantifies differences in recombination rate between the sexes in outbred populations. It has the potential to integrate with cytogenetic and other physical maps, identifying paralogous regions of the rainbow trout genome arising from the evolutionarily recent genome duplication event, and anchoring a comparative map with the zebrafish, medaka, tetraodon, and fugu genomes. This resource will facilitate the identification of genes affecting traits of interest through fine mapping and positional cloning of candidate genes.

Project description:BackgroundMaternal transcripts are accumulated in the oocyte during oogenesis to provide for protein synthesis from oocyte maturation through early embryonic development, when nuclear transcription is silenced. The maternal mRNAs have short poly(A) tails after undergoing post-transcriptional processing necessary for stabilizing them for storage. The transcripts undergo cytoplasmic polyadenylation when they are to be translated. Transcriptome analyses comparing total mRNA and elongated poly(A) mRNA content among eggs of different quality can provide insight into molecular mechanisms affecting egg developmental competence in rainbow trout. The present study used RNA-seq to compare transcriptomes of unfertilized eggs of rainbow trout females yielding different eyeing rates, following rRNA removal and poly(A) retention for construction of the libraries.ResultsThe percentage of embryos to reach the 32-cell stage at 24 h post fertilization was significantly correlated to family eyeing rate, indicating that inviable embryos were developmentally compromised before zygotic genome activation. RNA sequencing identified 2 differentially expressed transcripts (DETs) from total mRNA sequencing comparing females with low-quality (< 5% eyeing), medium-quality (30-50% eyeing), and high-quality (> 80% eyeing) eggs. In contrast, RNA sequencing from poly(A) captured transcripts identified 945 DETs between low- and high-quality eggs, 1012 between low- and medium-quality eggs, and only 2 between medium- and high-quality eggs. The transcripts of mitochondrial genes were enriched with polyadenylated transcript sequencing and they were significantly reduced in low-quality eggs. Similarly, mitochondrial DNA was reduced in low-quality eggs compared with medium- and high-quality eggs. The functional gene analysis classified the 945 DETs between low- and high-quality eggs into 31 functional modules, many of which were related to ribosomal and mitochondrial functions. Other modules involved transcription, translation, cell division, apoptosis, and immune responses.ConclusionsOur results indicate that differences in egg quality may be derived from differences in maternal nuclear transcript activation and cytoplasmic polyadenylation before ovulation, as opposed to accumulation and storage of maternal nuclear transcripts during oogenesis. Transcriptome comparisons suggest low-quality eggs suffered from impaired oxidative phosphorylation and translation. The DETs identified in this study provide insight into developmental competence in rainbow trout eggs.

Project description:The sea-run phenotype of rainbow trout (Oncorhynchus mykiss), like other anadromous salmonids, present a juvenile stage fully adapted to life in freshwater known as parr. Development in freshwater is followed by the smolt stage, where preadaptations needed for seawater life are developed making fish ready to migrate to the ocean, after which event they become post-smolts. While these three life stages have been studied using a variety of approaches, proteomics has never been used for such purpose. The present study characterised the blood plasma proteome of parr, smolt and post-smolt rainbow trout using a gel electrophoresis liquid chromatography tandem mass spectrometry approach alone or in combination with low-abundant protein enrichment technology (combinatorial peptide ligand library). In total, 1,822 proteins were quantified, 17.95% of them being detected only in plasma post enrichment. Across all life stages, the most abundant proteins were ankyrin-2, DNA primase large subunit, actin, serum albumin, apolipoproteins, hemoglobin subunits, hemopexin-like proteins and complement C3. When comparing the different life stages, 17 proteins involved in mechanisms to cope with hyperosmotic stress and retinal changes, as well as the downregulation of nonessential processes in smolts, were significantly different between parr and smolt samples. On the other hand, 11 proteins related to increased growth in post-smolts, and also related to coping with hyperosmotic stress and to retinal changes, were significantly different between smolt and post-smolt samples. Overall, this study presents a series of proteins with the potential to complement current seawater-readiness assessment tests in rainbow trout, which can be measured non-lethally in an easily accessible biofluid. Furthermore, this study represents a first in-depth characterisation of the rainbow trout blood plasma proteome, having considered three life stages of the fish and used both fractionation alone or in combination with enrichment methods to increase protein detection.

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Endoplasmic reticulum stress as a key mechanism in stunted growth of seawater rainbow trout (Oncorhynchus mykiss)

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