Introduction
A growing number of consumers in Europe prefer aquaculture fish on the plate that had the "feeling" of a "free and happy" life. Beside of many other factors, this stands for fish that is produced under optimal rearing conditions without drugs against infections, temperature stress, or not acceptable stocking rates. One of the common characteristics of the German rainbow trout aquaculture is that all-female eggs are imported from worldwide acting distributors and then hatched and bred. The mixture of genetically non-adapted rainbow trout (Oncorhynchus mykiss) to the rearing region and in many cases suboptimal local aquaculture conditions results therefore in higher mortalities and slower growth performance, which in turn may cause high economic losses to the farmer. An open question in this context concerns fish welfare. An alternative would be the selection and breeding of robust local rainbow trout strains that are genetically adapted to regional environmental stressors. Aim of our studies is therefore to determine and comprehensively characterize the trait robustness in rainbow trout. This includes holistic and comparative molecular analyses of unstressed and stressed trout of an imported and a regional trout strain and the identification of genes putatively responsible for stress and immune response. Such molecules could be exploited as biomarkers that contribute to the development of a simple molecular test tool for measurement of stress in rainbow trout aquaculture. Furthermore, the local strain will serve as fish model for the identification of DNA polymorphisms potentially associated with the trait robustness.
Materials and methods
In all experiments, we used the rainbow trout strain BORN (Born, Germany) selected for survival under regional conditions since 1975 (Anders, 1986; Rebl et al., 2014) and in comparison to it all-female rainbow trout selected for growth (Steelhead II-WA; Troutlodge, Tacoma, USA). Beside of phenotype and immunological analyses, we performed transcriptome studies using different types of microarrays (16K cDNA cGRASP chip, von Schalburg, 2008; 4x44K and 8x60K Salmon Agilent 60-mer oligo chips) and a paired-end sequencing approach (Illumina). The experimental approach included the comparison of healthy not affected trout and trout stressed by challenge experiments such as moderate temperature displacement to 8°C or 23°C, i.p. infection with Aeromonas salmonicida, or crowding stress. All experiments followed scientific (e.g. exclusion of tank effects; use of biological replica) and animal welfare criteria (e.g. monitoring of environmental factors; anesthesia of fish in 50 ng/mL benzocaine solution. We used well-standardized procedures for sampling, tissue conservation and preparation of tissue-specific RNA. Microarray experiments were performed as one-color (Agilent chips) or two color (cGRASP chip) experiments. Performing standardized normalization, statistical tests (including Bonferoni correction and Principal Component Analyses - PCA), calculation of gene expression and fold-change values as well as gene annotation identified significant (FC ≥ 2) differences in gene expression. STARS bioinformatics package and Ingenuity Pathway Analyses (IPA) classified genes by their functions. Quantitative real time PCR (qPCR) using Roche Lightcycler96 or the 192.24 IFCs for the Fluidigm Biomark HD system validated selected microarray results. Procedures are described in more detail by e.g. Rebl et al. (2013, 2014), Köbis et al. (2013, 2015), and Borchel (2015).
Results and discussion
In focus of our research was first, if the imported rainbow trout selected for growth grow better than the regional strain selected for survival. Indeed, under optimal environmental conditions the imported strain grows significantly better. However, after temperature stress (27°C) occurred in an open pond facility, we assigned better growth performance to the regional strain (weight gain +60%, 150g). The comparison of innate immunity parameters of unstressed healthy trout revealed increased expression of protective genes in liver of BORN trout, which might contribute to an improved first barrier against pathogen invasion (Rebl et al., 2012). Furthermore, the functional characterization of complement genes in unstressed fish revealed a down-regulation of numerous genes in BORN trout; e.g. C4, C1QL4, CFD (Köbis et al., 2015). The differences compared to the imported trout indicate a more efficient innate immune system in BORN trout. Experimental temperature stress resulted in strain-specific transcriptome regulation of well-known heat/cold stress genes (e.g. SERPINH1, CIRBP). In addition, we detected several novel genes correlated to thermoregulation; e.g. UCHL1, CYP1C1 (Rebl et al., 2014). Numerous of these genes belong to the acute-phase. Experimental infection of trout with Aeromonas salmonicida resulted in higher survival rates in strain BORN. Transcriptome analysis of genes of peritoneal leucocytes 12h and 72h after infection identified multiple expression differences of pathogen response molecules and cytokines between strains indicating different defense strategies of trout strains (Korytar et al., 2013). The evaluation of all performed transcriptome analyses resulted in a small number of genes that are constantly differently expressed between the compared trout strains; e.g. GIMAP7 (Borchel et al., 2015). We suggest the identified strain-specificly regulated genes as candidate genes describing the trait robustness. Genes similarly regulated in both strains but differently after stressor impact indicate genes that could potentially serve as general stress markers in rainbow trout aquaculture.
Funding
This work was supported by the European Fisheries Fund and the Ministry of Agriculture, the Environment and Consumer Protection Mecklenburg-Western Pomerania (pilot project BORN trout # VI 560 7308-4).
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