Location: Cool and Cold Water Aquaculture Research
2024 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
Progress toward Sub-objective 1a: Fillet yield phenotyping of fourth-generation nucleus families from the ARS-FY-H (n = 98), ARS-FY-L (n = 22), and ARS-FY-C (n = 30) genetic lines was completed, and mean fillet yields were 58.0% (standard error = 0.13), 54.9% (standard error = 0.27), and 55.4% (standard error = 0.47), respectively. Compared to the ARS-FY-C (unselected control) line, 4 generations of selection has resulted in an upward phenotypic selection response in the ARS-FY-H line of +0.67 (standard error = 0.10; P = 0.003) percentage points per generation and a downward phenotypic selection response in the ARS-FY-L line of -0.24 (standard error = 0.11; P = 0.09) per generation. The increased fillet yield in the ARS-FY-H line is primarily due to a reduction in viscera yield (i.e., fat accumulation in the visceral cavity); correlated phenotypic selection response for viscera yield in the ARS-FY-H line is -0.55 (standard error = 0.02; P < 0.0001) percentage points per generation. Growth performance (i.e., body weight at harvest) has not been affected by fillet yield selection in the ARS-FY-H line (P = 0.91). Broodstock from the fourth-generation nucleus families were selected and spawned to produce fifth-generation nucleus families for the ARS-FY-H (n = 100), ARS-FY-L (n = 23), and ARS-FY-C (n = 28) genetic lines. Fish from these families will be PIT-tagged beginning in July 2024 for grow-out in communal tanks.
Progress toward Sub-objective 1c: Bacterial cold water disease (BCWD) causes significant economic losses in rainbow trout, and selection for BCWD resistance is one of the major goals of commercial aquaculture breeding programs. Based on sequence analysis of pooled DNA samples, two chromosome segments that span the disease resistance genes were identified. A shortlist of candidate genes for BCWD resistance was determined after examination of the predicted function of all the genes located in those two chromosome segments. Rainbow trout families with or without the favorable QTL genotype were phenotyped for resistance to BCWD disease last fall, and samples were collected for RNA sequencing and gene expression analysis. Over 30 additional crosses with known QTL genotypes were made this spring and will be further evaluated for BCWD resistance in the fall. This germplasm will be used for further functional evaluation of the candidate genes for BCWD resistance. Additionally, rainbow trout cell lines including the RTG-2, RTG-P1, RTgill-W1, RTH-149 and RTS-11 were acquired and adapted to different cell culture media conducive to electroporation conditions. These cell lines will be used to evaluate functional mechanisms of genes relevant to BCWD using gene editing technologies. Expression of candidate genes within QTLs have been confirmed in transcriptomic datasets for the RTS-11 and in vitro functional assays have been optimized. Guide RNAs and molecular tools to determine editing efficiency have been designed for CRISPR/Cas9 gene editing efforts.
Progress toward Sub-objective 1d: A study was performed to identify genomic regions with selection signatures in the USDA rainbow trout broodstock population that was selected over five generations for resistance to bacterial cold water disease (BCWD). Whole genome sequencing and genotype imputation were used to identify approximately nine million single nucleotide polymorphism (SNP) markers in the population and those genotypes were used to identify chromosome regions with selection signatures within and between the genetic lines. Overall, 17 genomic regions were identified with significant pattern of positive selection signature, of which three overlapped with previously found QTL for BCWD resistance in the USDA rainbow trout breeding program.
Progress toward Sub-objective 1e: Whole genome de-novo assemblies of four cutthroat trout genomes were completed. Currently we are working with collaborators to generate genetic linkage maps that will be used to guide chromosome level sequences for the cutthroat trout genomes. The genomes from the Bonneville, Yellowstone, West slope, and Coastal cutthroat trout lineages represent a large component of the geographic distribution and genetic diversity of this salmonid species. Cutthroat trout has great ecological, cultural, and economic importance for indigenous communities and for the sport fishing and the tourism industry in the Pacific Northwest.
Progress toward Sub-objective 2b: Two independent populations of rainbow trout were produced through breeding gene edited F0 fish. Offspring were homozygous mutants with targeted disruption of either insulin-like growth factor-2b (IGFBP-2b) or lysosome-associated membrane protein-2A (lamp2a). Two studies were performed to analyze the loss-of-function phenotype with emphasis on growth performance and nutrient partitioning. Compared to controls, fish lacking a functional IGFBP-2b exhibited increased appetite, faster growth, and better feed efficiency during satiable feed intake, but responded similarly to feed deprivation. Subsequent protein and gene expression analysis indicated compensatory response of IGF-system components, including reduced serum IGF-1 and differential expression of other IGFBPs. Findings provide support for a role of IGFBP-2b in regulating IGF-1 half-life in circulation and anabolic mechanisms directing somatic tissue growth. The second study evaluated how loss of functional LAMP2A impacted growth performance and nutrient partitioning during consumption of a high carbohydrate diet. Fish lacking LAMP2A grew better, exhibited biomarkers of higher feed intake, and displayed a unique hepatic proteome supporting differences in amino acid, lipid, and glucose metabolism. LAMP2A knockout fish also had disruption of oxidative stress regulatory mechanisms and may exhibit greater susceptibility to Flavobacterium psychrophilum challenge. Findings support that LAMP2A plays an essential role in chaperone mediated authophagy, which regulates nutrient metabolism and oxidative stress, thereby characterizing a central mechanism regulating the balance between anabolic and catabolic responses.
Progress toward Sub-objective 2d. The samples from the water quality study were lost in a -80C freezer malfunction as reported last year. Samples from the study on the effects of overripening on egg quality were sent for PAIso-Seq sequencing last year and results were obtained this year. The study consisted of eggs from three stages from each of six fish. Five of the samples including three from the same fish failed to yield useable data and therefore new libraries including for three samples from an additional fish had to be constructed and sequenced, delaying analysis. Over 2.5 million sequences were obtained for each sample. Our previous work suggests egg quality is dependent upon proper activation of maternal transcripts stored in the egg. Transcripts are stored with short poly(A) tails that are then elongated upon activation for translation. We did not find a transcriptome-wide difference in mean tail length or a skewing of tail length from short to long with activation in response to fertilization. This suggests transcriptome-wide changes in transcript tail-length profiles cannot be used as metric for egg quality. Changes in tail-length of specific differentially expressed genes (DEGs) may better serve as markers.
Accomplishments
1. Generation of an improved reference genome for rainbow trout. 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 years, ARS researchers in Leetown, West Virginia, have significantly improved the quality and annotation of the rainbow trout reference genome from the Arlee genetic line of rainbow trout. In addition, they completed and released three additional high quality de-novo genome assemblies from rainbow trout lines that represent wide genetic diversity and geographic distribution. The importance of the improved reference genome has been demonstrated by its wide adoption and use in genetics and physiology in rainbow trout.
2. Characterizing genetic regulation of IHNV disease resistance in rainbow trout. Infectious hematopoietic necrosis (IHN) is a disease of salmonid fish that is caused by the IHN virus (IHNV). Under intensive aquaculture conditions, IHNV can cause significant mortality and economic losses. Currently, there is no proven and cost-effective method for IHNV control. In collaborative research with multiple institutions and industry, ARS researchers in Leetown, West Virginia, found that genetic resistance to IHNV is controlled by the inheritance of several moderate-effect quantitative trait loci (QTL) and many small effect loci in three 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 resistance to IHN disease in commercial rainbow trout populations. Several candidate genes were identified, and further foundational research on how these genes are involved in antiviral response in rainbow trout will improve our understanding of the biological basis of resistance to this devastating viral disease.
3. Characterizing the functional role of insulin-like growth factor binding protein-2b (IGFBP-2b) in rainbow trout. Insulin-like growth factor-1 (IGF-1) is a hormone that promotes body growth, primarily through support of muscle and bone development. In rainbow trout, 99% of IGF-1 circulating in blood is bound to IGFBPs, the most abundant of which is IGFBP-2b, however the functional significance of this binding protein as a regulator of IGF signaling in fish has not been characterized. Through gene editing and breeding, researchers in Leetown, West Virginia, generated a line of rainbow trout lacking a functional IGFBP-2b. Despite exhibiting a 25% reduction in IGF-1 in blood, these fish ate more and grew faster than controls. Fish without IGFBP-2b also displayed unique expression levels of other IGFBP genes and proteins, supporting that a compensatory response occurred to maintain appropriate levels of IGF-1 signaling. Findings suggest that IGFBP-2b has a role in extending the half-life of circulating IGF-1, and functional redundancy exists between iGFBP-2b and other IGFBP family members. These results provide insight into physiological mechanisms regulating growth traits in rainbow trout.
Review Publications
Vallejo, R.L., Pietrak, M.R., Milligan, M.T., Gao, G., Tsuruta, S., Fragomeni, B.O., Long, R., Peterson, B.C., Palti, Y. 2024. Genetic architecture and accuracy of predicted genomic breeding values for sea lice resistance in the St John River aquaculture strain of North American Atlantic salmon. Aquaculture. 586:740819. https://doi.org/10.1016/j.aquaculture.2024.740819.
Freij, K., Cleveland, B.M., Biga, P. 2024. Maternal dietary choline levels cause transcriptome shift due to genotype-by-diet interactions in Rainbow Trout (Oncorhynchus mykiss). Comparative Biochemistry and Physiology, Part D: Genomics and Proteomics. 49:101193. https://doi.org/10.1016/j.cbd.2024.101193.
Leet, J.K., Richter, C.A., Claunch, R.A., Gale, R.W., Tilitt, D.E., Iwanowicz, L.R. 2024. Immunomodulation in adult largemouth bass (Micropterus salmoides) exposed to a model estrogen or mixture of endocrine disrupting contaminants during early gonadal recrudescence. Comparative Immunology Reports. 6:200140. https://doi.org/10.1016/j.cirep.2024.200140.
Weber, G.M. 2023. Effects of IGF1 on in vitro ovarian follicle maturation in rainbow trout (Oncorhynchus mykiss). Fishes. 8(7):367. https://doi.org/10.3390/fishes8070367.
Raines, C.D., Iwanowicz, L.R., Lovy, J., Phelps, N., Mor, S., Ng, T. 2024. Discovery and genomic characterization of a novel metahepadnavirus from clinically normal anadromous alewives (Alosa pseudoharengus). Viruses. 16(6):824. https://doi.org/10.3390/v16060824.
Palti, Y., Vallejo, R.L., Purcell, M., Gao, G., Shewbridge, K., Long, R., Setzke, C., Fragomeni, B., Cheng, H., Martin, K., Naish, K. 2024. Genome-wide association analysis of the resistance to infectious hematopoietic necrosis virus in two aquaculture rainbow trout strains confirms oligogenic architecture with several moderate effect quantitative trait loci. Frontiers in Genetics. 15:1394656. https://doi.org/10.3389/fgene.2024.1394656.
Lovy, J., Iwanowicz, L.R., Welch, T.J., Allam, B., Getchell, R., Geraci-Yee, S., Good, C., Snyder, J., Raines, C., Das, N. 2024. Seasonal mortality of Atlantic menhaden is associated with neurologic disease caused by a virulent clone of Vibrio anguillarum: Fish kills and implications for biosecurity. Transboundary and Emerging Diseases. 18:8816604. https://doi.org/10.1155/2024/8816604.
Ahmed, R.O., Ali, A., Leeds, T.D., Salem, M. 2023. RNA-Seq analysis of the pyloric caecum, liver, and muscle reveals molecular mechanisms regulating fillet color in rainbow trout. BMC Genomics. 24:579. https://doi.org/10.1186/s12864-023-09688-5.