Project : USDA ARS

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Research Project: Integrated Research Approaches for Improving Production Efficiency in Rainbow Trout

Location: Cool and Cold Water Aquaculture Research

2025 Annual Report

Objectives
Objective 1: Improve performance of aquaculture production traits in rainbow trout by developing enhanced selective breeding strategies and genomic technologies: 1a: Selective breeding, evaluation of genomic selection, and development of improved germplasm with superior fillet yield; 1b: Analysis of the genetic architecture and evaluation of the accuracy of genomic selection for resistance to infectious hematopoietic necrosis virus (IHNV) in commercial rainbow trout breeding populations; 1c: Identification of candidate genes for bacterial cold water disease (BCWD) resistance in rainbow trout using pool-seq and improvement of marker-assisted selection for BCWD resistance in multiple rainbow trout breeding populations; 1d: Detection and characterization of genomic signature and selective sweeps associated with phenotypic selection for improved resistance to BCWD in rainbow trout; and 1e: Improvement of the rainbow trout reference genome assembly and analysis of structural variations. Objective 2: Characterization of reproductive and metabolic mechanisms affecting production traits to better define phenotypes and improve selective breeding and management practices: 2a: Characterize attributes of fillet quality and feed utilization efficiency in rainbow trout selectively bred for divergent fillet yield phenotypes; 2b: Utilize gene editing technology to better understand and improve growth performance and nutrient utilization; 2c: Characterization of maternal transcript processing; and 2d: Identification of molecular markers for changes in egg quality in response to hatchery conditions and practices.

Approach
Rainbow trout (Oncorhynchus mykiss) are farmed in over half of US states and represent the second most valuable domestic finfish aquaculture product. Although production has increased, the US still imports approximately 50% of the rainbow trout sold for food, so the potential exists to increase domestic production to meet current demand. Increasing production efficiency, product quality, and fish health is central for industry expansion. This project contributes to industry expansion by integrating genomic technologies and enhanced phenotypes with selective breeding strategies that maximize genetic improvements in fillet yield, disease resistance, and reproductive success. Previously, NCCCWA scientists determined that integrating genomic selection with conventional breeding strategies improved genetic gains for resistance to bacterial cold water disease. This project aims to 1) refine genomic selection protocols to support commercial implementation of this breeding technology and 2) develop and evaluate genomic selection tools to (independently) increase fillet yield and improve resistance to infectious hematopoietic necrosis and bacterial cold water disease. Accompanying selective breeding for fillet yield will be an analysis of economically important traits such as growth, feed efficiency, and fillet quality to determine whether selection has indirect effects on performance, nutrient utilization, and product quality. Using gene editing and functional genomics to investigate the physiological mechanisms regulating nutrient metabolism and egg quality will better define these phenotypes, improve understanding of their response to selective breeding, and identify husbandry strategies that optimize performance. Collectively, this project will provide the rainbow trout industry with improved germplasm, genomic selection technologies to accelerate genetic gains, and physiological insights towards improving fish culture.

Progress Report
Sub-objective 1a: Upon completion of this project, four generations of divergent selection for fillet yield were conducted resulting in development of high (ARS-FY-H), low (ARS-FY-L), and randomly-mated control (ARS-FY-C) genetic lines. Including the base population and all four generations of selection, a total of 397 ARS-FY-H, 91 ARS-FY-L, and 213 ARS-FY-C nucleus families (3,234 fish total) were reared to a market body weight of 1.2 to 1.9 kg and characterized for fillet yield. Analysis of selection response data through 4 generations of selection demonstrated that fillet yield was heritable (0.48, SE = 0.04) and negatively correlated (-0.60, SE = 0.05) with viscera yield. Compared to the randomly-mated ARS-FY-C line, fillet yield increased in the ARS-FY-H line by 0.67 (SE = 0.10) percentage points per generation due to upward selection and decreased in the ARS-FY-L line by 0.24 (SE = 0.11) percentage points per generation due to downward selection. Fourth-generation ARS-FY-H fish had greater condition factor (1.50, SE = 0.01) and body weight (1,912 grams, SE = 19.7 grams) compared to ARS-FY- L (1.37, SE = 0.02; 1,840 grams, SE = 41.4 grams, respectively) fish. The increased fillet yield in the ARS-FY-H line is primarily attributable to less deposition of fat in the viscera as viscera yield decreased by 0.55 (SE = 0.02) percentage points per generation, and there were no adverse effects of fillet yield selection on fillet texture. In addition, using genome-enabled approaches for selective breeding holds great potential for further improving the accuracy of animal genetic merit predictions. In a retrospective evaluation using fillet yield data from four generations the genomic-enabled approach was shown to improve the prediction accuracy by 50%. Relation to the new project plan: The National Center for Cool and Cold Water (NCCCWA) selective breeding program will continue the 5th and final year of selection for fillet yield . Sub-objective 1b: In collaborative research with multiple research institutions and industry, we found that genetic resistance to infectious haematopoietic necrosis virus (IHNV) is controlled by the oligogenic inheritance of several moderate effect quantitative trait loci (QTL) and many small effect loci in three different commercial rainbow trout breeding populations that are widely used by the U.S. aquaculture industry. The trait was found to be moderately heritable, indicating that selective breeding can be used to improve the genetic basis for resistance to IHNV disease in the commercial rainbow trout populations. Sub-objective 1c: Bacterial cold-water disease (BCWD) is one of the most devastating diseases in rainbow trout aquaculture. Improving resistance to BCWD using traditional family-based selective breeding has shown promise but is limited since it requires repeated disease trials for every generation and the resistance cannot be measured directly in potential breeders. Here we identified a set of six DNA markers that can be used to predict the genetic merit for resistance to BCWD of potential breeding animals with the same or better accuracy than the traditional family-based selective breeding approach and without the need to repeat the disease trial in consecutive generations. The set of genetic markers we identified has been incorporated into the selective breeding scheme of the U.S. rainbow trout aquaculture industry’s largest eyed egg producer with positive feedback from fish farmers. Relation to the new project plan: In the new plan we will conduct transcriptome and gene expression analysis with the aim of identifying the causative genes for resistance to BCWD that are residing near the set of DNA markers that were identified. Sub-objective 1d: A study was performed to identify genomic regions with selection signatures in the USDA-ARS rainbow trout broodstock population that was selected over five generations for resistance to bacterial cold water disease (BCWD). Overall, 17 genomic regions were identified with significant pattern of positive selection signature, of which three overlapped with previously found quantitative trait loci (QTL) for BCWD resistance in the USDA rainbow trout breeding program. This study demonstrated that the selective sweep analysis approach was not effective in enriching for genome regions and candidate genes that are associated with selection for BCWD resistance. Sub-objective 1e: A high-quality reference physical genome map is important for facilitating meaningful genetic analyses and enhancing research on the physiology of the organism. In the past 5-year project cycle we significantly improved the quality and annotation of the rainbow trout reference genome from the Arlee doubled haploid (DH) clonal line of rainbow trout. In addition, we completed and released three additional high quality de- novo genome assemblies from rainbow trout lines that represent wide genetic diversity and geographic distribution. Relation to the new project plan: In the new plan we will annotate the coding genes on the four rainbow trout genome maps that we generated in the previous project with the aim of providing a more comprehensive pan- genome reference that will capture most of the genic diversity in rainbow trout. Genomic structural variants (SVs) refer to changes in the length or orientation of the DNA sequence at specific locations in the genome. Whole-genome sequence data from 96 fish was used to identify 13,863 SVs in the genome of farmed rainbow trout from three U.S. breeding programs. This was a first- of- its- kind study in rainbow trout providing the foundation for studying the role of this important and more abundant than previously thought source of genomic and genetic variation. Relation to new project plan: Additional characterization and cataloging of structural variants using long-reads sequencing and a larger sample size that will provide better representation of the genetic diversity in U.S. rainbow trout aquaculture is proposed in the new project plan. Sub-objective 2a: Two studies were performed to characterize indirect effects of selection for fillet yield on economically important phenotypes associated with growth performance, product quality, and nutrient utilization. First, the High Yield (HY) and Low Yield (LY) lines were analyzed for differences in feed conversion ratio (FCR). Findings indicated there was no significant difference in FCR, supporting that selection for improved fillet yield did not affect how efficiently food is converted into biomass. A second study determined whether increased fillet yield in the HY line was dependent on dietary formulation. Across all diets, the HY line produced significantly higher percent fillet yield, indicating that producers of rainbow trout bred for high fillet yield do not have to change their current nutritional strategy to achieve improved product yields. Also important was that selection for high fillet yield did not have negative consequences on fillet quality. Subobjective 2b: Utilize gene editing technology to better understand and improve growth performance and nutrient utilization. Two independent lines of rainbow trout were produced with disruptions in either a functional 1) insulin- like growth factor-2b (IGFBP-2b) or 2) lysosome-associated membrane protein-2b (LAMP-2a). Analysis of a homozygous F2 generation of IGFBP-2b knockouts (2bKO) indicated increased appetite and faster growth rate compared to wild type fish, despite exhibiting a 20-50% reduction in plasma insulin-like growth factor-1 (IGF1). Phenotyping the second line of rainbow trout with LAMP-2a knockout (Chr 31 only) showed increased growth and tolerance of a high-carbohydrate containing diet in these fish compared to wild types. Gene expression and proteomic analysis indicated regulation of glycolytic mechanisms in the knockout fish, providing evidence that LAMP-2a and chaperone-mediated autophagy are important for maintaining nutritional homeostasis in rainbow trout. Additional phenotyping studies are planned in the next Project (8082-10600-002-000D) to define the phenotype of rainbow trout with complete loss of LAMP-2a on both Chr 14 and 31. Sub-objective 2C. The processing of mRNA transcripts in the oocyte is very different from that of most cells and involves complex regulation of the mRNAs’ polyadenylate (polyA) tail. Procedures for global measurement of mRNA polyA tail lengths are new and rapidly evolving. We evaluated several approaches and determined PAIso- seq was the best method for cost-effective analysis. We then compared global mRNA profiles of 1) unfertilized eggs which is before most of the stored maternal mRNA is activated, 2) eggs 24hrs after fertilization which is when many of the maternal transcripts are being or have been translated into proteins, and 3) 5 days after fertilization when the embryo is dependent on embryonic transcripts. Overall differences in polyA tail lengths among the stages were less than expected and we are now comparing genes belonging to different pathways associated with key developmental processes along the progression from unfertilized egg to mid-stage embryo (Project #8082- 10600-002-000D). Sub-objective 2D. We evaluated the effect of post-ovulatory aging on egg quality and found egg quality measured as eying rate rapidly decreased over time. PAIso-seq analysis of samples from a post-ovulatory aging time series was conducted. Many differentially expressed genes were identified between freshly ovulated eggs and eggs that were 14 days post-ovulation that may serve as markers of not only egg quality, but indicators a hatchery may have a problem with post-ovulatory aging and therefore need to check their broodstock for ovulation more often. The overall polyA tail lengths of transcripts did not change over time, but changes in tail lengths of transcripts differentially expressed with days post-ovulation will be assessed in Project #8082-10600-002-000D.

Accomplishments
1. Defined the physiology driving high fillet yield in rainbow trout. ARS scientists in Leetown, West Virginia, used selective breeding to produce a line of rainbow trout exhibiting high fillet yield (ARS-FY-H). Although the fillet yield trait is well-defined, the physiological mechanisms driving increased muscle mass in the High Yield line are unknown. Therefore, the muscle and liver transcriptome were analyzed to identify differentially expressed genes between the High Yield and a Low Yield line. Findings indicate that greater muscle growth in the High Yield line is driven by higher rates of protein accretion caused by reductions in protein degradation, increased muscle cell proliferation and differentiation, and more efficient use of nutrients. In the liver, gene expression signatures indicate the High Yield line is less obese, prioritizing nutrients for muscle growth rather than storage as visceral fat. These findings define the unique physiology of the High Yield line that result in greater muscle mass and contribute to development of breeding strategies towards genetic advancement for this economically important trait.

2. Characterizing interactions of insulin-like growth factors and a progestin in rainbow trout reproduction. Maturation inducing hormones (MIH) function to induce changes in the unfertilized egg in preparation for fertilization. Growth factors such as insulin-like growth factor (IGF) also participate in maturational changes in the unfertilized egg in many species. These hormones act on cells of the follicle which is the cell layer that surrounds the egg; however, the mechanism of hormone action on maturational changes is not well understood. Using in vitro tissue culture systems, ARS researchers in Leetown, West Virginia, have shown the rainbow trout to be unique among fishes in that IGFs are unable to induce changes in follicle cells or eggs that increase sensitivity to MIH or cause maturational progression. Nevertheless, in rainbow trout, MIH uses the same signaling pathways that are used by IGFs to induce egg maturation in other species. These findings demonstrate the species-specific complexity of hormonal regulation of maturation among fishes and suggest IGFs cannot serve as therapeutics to control spawning in rainbow trout.

3. Characterizing the transcriptomic response of the rainbow trout ovary to a progestin. In most fishes, including the rainbow trout, maturation inducing hormones (MIH) induce changes in the unfertilized egg in preparation for fertilization. Egg maturation involves many complex changes yet the egg has little if any capacity for gene expression and therefore must rely on activation of maternal-derived gene transcripts to support progression through maturation. However, the physiological mechanisms associated with maturational progression are not well understood. ARS researchers in Leetown, West Virginia, used in vitro tissue culture and comprehensive gene expression analysis to characterize changes in the transcriptome of the follicle-egg complex to unravel the processes involved in MIH-induced egg maturation. Among the many physiological pathways that responded to MIH, transcripts associated with signal transduction pathways were the most extensive. These findings support a pervasive influence of MIH on actions of other growth factors in the follicle-egg complex that together contribute to the successful maturation of the oocyte into a fertilizable egg. Results contribute to the development of management strategies that improve reproductive efficiency in rainbow trout.

U.S. DEPARTMENT OF AGRICULTURE

Cool and Cold Water Aquaculture Research: Leetown, WV