Research Project: Reducing Impacts of Disease on Salmonid Aquaculture Production

Location: Office of The Director

2018 Annual Report

Objectives
Major impediments to production and profitability of U.S. aquaculture are the lack of genetically-defined species with traits for faster growth, greater feed efficiency/utilization, and improved disease resistance. Rainbow trout are important recreational and food fish species in the Great Lakes and it is thus important to improve productivity of this species in this region. Over these next 3 years we will focus on the following three Objectives and their supporting Sub-Objectives: Objective 1: Characterize mechanisms of innate immune response, and pathogen virulence, to control rhabdoviral diseases in salmonid aquaculture. • Sub-Objective 1.A.: Identify domains within viral proteins of infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) that interfere with the host virus recognition and response pathways in vitro. (Leaman, Stepien and Vakharia) • Sub-Objective 1.B.: Characterize the in vitro replication of recombinant IHNV and VHSV containing mutations designed to disrupt viral suppression of host recognition and response pathways. (Leaman and Vakharia) • Sub-Objective 1.C.: Assess the impact of IHNV and VHSV infection and subsequent innate immune suppression on the activation of dendritic cells (DCs). (Spear) • Sub-Objective 1.D.: Develop in vivo IHNV and VHSV challenge models in rainbow trout. (Spear and Shepherd) Objective 2: Use genetic techniques to characterize mechanisms of Flavobacterium virulence and identify potential strategies to control bacterial disease in salmonid aquaculture. • Sub-objective 2.A.: Develop genetic techniques for F. columnare strains that cause columnaris disease in rainbow trout. (McBride) • Sub-Objective 2.B.: Isolate and characterize F. columnare mutants and identify virulence factors associated with ability to cause disease in rainbow trout. (McBride) • Sub-Objective 2.C.: Develop improved genetic techniques for F. psychrophilum, the causative agent of bacterial coldwater disease. (McBride) Objective 3: Measure and modulate antimicrobial peptides (AMPs) as a means to control disease in salmonids. • Sub-Objective 3.A.: Characterize the environmental and endocrine contributions to regulation and expression of AMPs in rainbow trout. (Shepherd and Spear) • Sub-Objective 3.B.: Test the anti-viral and anti-bacterial activities of two synthetic trout AMPs in vitro. (Shepherd, Spear, Leaman and McBride)

Approach
For Objective 1: We will characterize the mechanisms of virulence for Viral Hemorrhagic Septicemia virus (VHSV) and Infectious Hematopoietic Necrosis virus (IHNV) in rainbow trout. Research will involve molecular analysis of viral diversity, mutational analysis of viral factors contributing to virulence in rainbow trout. These studies will utilize homologous in vitro systems (cell-lines and dendritic cells) to identify of host factors involved in recognition and response pathways to viral infection in rainbow trout. Lastly, disease challenge assays will be developed, and validated, to understand the virulence and the disease processes for IHNV and VHSV pathogens in rainbow trout. For Objective 2: This work will target mechanisms of pathogenesis of F. psychrophilum (causative agent in bacterial cold water disease) and F. columnare (causative agent in columnaris disease) in rainbow trout. To do this, we will use bacterial culture and genetic techniques to isolate mechanisms of pathogensis for Flavobacterium spp. in this species. Attenuated bacterial strains will be evaluated for pathogenesis using established disease challenge models, in this species. For Objective 3: We will characterize the physiological regulation of antimicrobial peptides (AMPs) and their actions in rainbow trout. To accomplish this, we will assess how environmental stressors, and hormones, affect expression (genes) and levels (proteins) of AMPs in this species. Additionally, we shall utilize in vitro techniques to evaluate biocidal actions of select AMPs against VHSV and IHNV and Flavobacterium spp.

Progress Report
For Objective 1, which is to understand the host interactions with the VHSV and IHNV pathogens, IHNV gene products have been cloned and expressed in fish cell-lines to determine their impact on the cellular innate immune responses. In fish cell-lines, results generally mirrored the effects of VHSV genes, with the exception of the IHNV N-gene, which had potent anti-host effects not seen in VHSV N-gene. IHNV M protein was still the most potent inhibitor of gene induction in cell-lines. To make the work more relevant to rainbow trout, research studies have transitioned to using two well-established cell-lines from rainbow trout, which are the RTG-2 (gonad) cells and RTgill-W1 cells. Results in trout cell-lines are comparable to the EPC (fathead minnow) cell-line, with some exceptions. Complementary efforts have also resulted in transition over to rainbow trout (rt) promoter constructs. Pursuing mutational studies, as a means to understand virulence in the IHNV pathogen, a series of mutations in IHNV M-gene have been constructed to mimic the D62A and E181A mutants made for VHSV M-gene. Using site directed mutagenesis, orthologous mutations (D63A, E178A and E189A) were made in a single mutation format with the IHNV M-gene. When comparing the response of different cell-lines to these mutagenesis products, the EPC cells behaved similarly to RTG-2 and RTgill-W1 cells. The D62A/E181A VHSV M-protein mutant was attenuated in all cells, suggesting that host proteins that engage mutated M-protein do so similarly in rainbow trout cells as compared to EPC cells. This is very interesting, and may provide a stronger means to identify host determinants of M-protein activity, which could enable development of attenuated therapeutics for many different fish species. As part of research efforts to develop improved viral challenge assays, a formulated dry diet was developed for juvenile lake sturgeon (Acipenser fulvescens). Given the resistance of lake sturgeon to the viral hemorrhagic septicemia virus (VHSV; a rhabdovirus) pathogen, and access to the newly formulated dry diet, this species can now be used as a negative control in viral challenge studies with other susceptible species that are also fed standardized dry diets. The use of a comparative approach, in disease challenge studies, will enable researchers to ask the question of how host (species) specificity/resistance occurs between economically important finfish species in the Great Lakes Region of the U.S. (e.g., salmonids, yellow perch and walleye). For Objective 2, which is to use genetic techniques to characterize mechanisms of Flavobacterium virulence and identify potential strategies to control bacterial disease in salmonid aquaculture, the MS-FC-4 F. columnare strain was selected as the primary pathogen model strain for studies of virulence, based on the ease with which this strain can be genetically manipulated. To facilitate work on F. columnare MS-FC-4 strain, the genome was sequenced and analyzed in year-2 and a paper was published on this topic. Further, several additional gene deletion mutations in this strain were constructed and efforts are underway to quantitatively analyze these deletion mutants. As expected, deletion of gldN, which encodes a component of the type IX secretion system (T9SS), resulted in loss of virulence. The results suggested that secreted proteins (toxic and non-toxic) are important for virulence and will impact considerations for production of vaccines as these toxins can negatively affect fish. Pursuing this idea further, Liquid Chromatography-Mass Spectroscopy (LCMS) was used to identify possible toxins that contribute to difficulties in creating a common vaccine for salmonid and non-salmonid fish species. As for secreted non-toxic proteins thought to be involved with virulence, genes encoding three secreted proteases and two secreted chondroitinases were identified and deleted. Single gene deletions had little effect on virulence against zebrafish, but a strain lacking all five of these genes exhibited greatly reduced virulence in zebrafish. Given species differences, similar studies will be conducted in rainbow trout to determine if this mutant is also deficient for virulence in a salmonid species. If it is, then efforts will focus on determining which of the five genes are most critical for virulence. If it is not deficient for virulence, additional (potential) virulence genes will be deleted to identify other critical virulence components. In this vein, another deletion mutant for sprF (a protein needed for secretion of the motility adhesin SprB to the cell surface) was constructed. This mutation nearly eliminates cell movement, and appears to result in reduced virulence on zebrafish. This deletion mutant will be tested using rainbow trout in the coming year. If either the motility deficient mutant or the mutants deficient for the enzymes listed above are avirulent for rainbow trout then we will determine if these strains can function as protective vaccines. Finally, fluorescently tagged versions of wild type and avirulent mutants of F. columnare, that have the gene encoding the green fluorescent protein inserted into the chromosome, have been constructed. These will be used to follow wild-type and avirulent mutant bacteria during infection challenge studies. Genetic studies of F. psychrophilum, the causative agent for cold-water disease in salmonids, have also been accomplished. In Year-1 a deletion mutant of F. psychrophilum THco2-90 strain (year-3 milestone), and its complemented mutant, were developed. In Year-2 these strains were analyzed for virulence to determine the importance of protein secretion in bacterial coldwater disease of rainbow trout in an injection challenge study. Whereas wild type and complemented strains were virulent for rainbow trout, the mutant was avirulent and thus failed to kill rainbow trout. These strains are being analyzed by international collaborators in France to examine the virulence of F. psychrophilum wild type and mutant cells in rainbow trout using an immersion challenge. For Objective 3, which is to measure and modulate antimicrobial peptides (AMPs) as a means to control disease in salmonids, a new family of AMPs have been identified in rainbow trout. Recently, 6 saposin-like proteins were discovered by this group. Bioinformatic analyses, using Big Data from the rainbow trout genome, have identified the chromosomal location of these genes. Structural modeling of the mature proteins show that they all contain the saposin-like tertiary fold/domain, placing them within the saposin family of proteins, specifically Nk-lysin protein sub-family. Initial transcriptomic data show that two are primarily expressed in the central nervous system and gonads, with another two being expressed in many tissues and the Nkl-like proteins being mostly expressed in immune-related tissues. To expand our understanding of how these AMPs are regulated, and verify the transcriptomic data, real-time quantitative PCR assays have been developed to measure gene expression for these AMPs in rainbow trout. Gene expression has been analyzed in tissues of Flavobacterium spp. challenged trout and in trout exposed to varying aquaculture stressors. To further study the importance and actions of these AMPs in rainbow trout, peptide cores have been designed, which are being commercially synthesized at this time. Once obtained, in vitro studies will be used to assess their bactericidal actions against two commercially-important bacterial pathogens, Flavobacterium columnare and Flavobacterium psychrophilum. Initially, collaborators have performed controlled experiments to monitor growth of F. columnare with small samples using the multi-well plate reader. While initial results results demonstrate these assays work, they do illustrate that solvent (e.g., Acetonitrile and Dimetylsulfoxide) levels must be held below 2% of the final assay volume as higher levels affect growth of the pathogen. Efforts from a previous research project (5090-31320-003-00D, Accession #0429302) have resulted in the sequencing and annotation of the yellow perch (Perca flavescens) genome. To accomplish this, genome sequencing coverage of over 79X was obtained using a long-read sequencing platform with an additional 20X coverage (a total of 99X coverage) from a short-read sequencing platform. Sequencing efforts resulted in 10,473 contigs with an N50 of 1.2M bp and a maximum contig size of 9.6M bp. Based on the raw long-read coverage of 79X, the yellow perch genome is estimated be 0.9 Gb in size and is comprised of ~ 31,500 genes. The long-read sequencing effort was polished with the 20X short-read genomic data and separate transcriptome data sets (4 separate tissue libraries) were used as evidence to validate the genome annotation. Initial analyses to assess the quality of the genome assembly, and annotation, was run via the Benchmarking Universal Single-Copy Orthologs (BUSCO) program. BUSCO analyses show that we have the following assessment results when compared with the “actinopterygii_odb9 gene set”: C:93.7%[S:86.2%,D:7.5%],F3.6%,M:2.7%,n:4584. Overall, the BUSCO results indicate a high quality genomic assembly, and annotation, for yellow perch. Gene scaffolds are being produced and the annotated genome will be mapped to these scaffolds. Once the scaffolding and annotation are complete, a novel RNA-seq data-set will be annotated to enable > 95% of reads to be mapped to a specific gene and scaffold with the aim of demonstrating the effects of gender on the immune response in this fish species.

Accomplishments
1. A new, formulated, dry diet improves growth in juvenile lake sturgeon. Sturgeon products (flesh and roe) are among the highest value commodities in global aquaculture. In the Midwest U.S., lake sturgeon (Acipenser fulvescens) are also a highly priced foof fish. However, our understanding of the necessary feeding rates, and access to formulated diets, is limited for this species. Such limitations currently result in feed waste and increased costs to producers. The primary aims of this work were to 1) develop a newly formulated dry diet for juvenile lake sturgeon, and 2) identify and compare optimal feeding rates of juvenile sturgeon fed the newly formulated dry diet against sturgeon fed a commercial semi-moist diet and live feeds (blood worms). ARS scientists in Milwaukee, Wisconsin in collaboration with researchers at the University of Wisconsin-Milwaukee showed that juvenile sturgeon fed the commercial semi-moist and the newly formulated dry diet grew faster than those fish fed the traditional live feeds. The optimal feeding rate was determined to be ~7.0% of body weight per day for juvenile (< 8 grams body weight) sturgeon. The use of a formulated dry diet, with an accurate feeding rate, will reduce waste, improve production efficiency and save aquaculture producers money.

2. Ghrelin (a gastric peptide) stimulates neuropeptides that control feed-intake, growth and immunity in fish. In fish, growth is generally related to feed intake. Small proteins, called neuropeptides, which are located in the brain and elsewhere, are known to affect all aspects of vertebrate physiology. Ghrelin is a peptide that is secreted by the stomach during fasting and signals the brain to increase feed intake. In addition, ghrelin has been shown to increase growth hormone levels and stimulate innate immunity in fish. However, our understanding of how ghrelin interacts within the brain to increase feed intake, growth and immune function is poor. ARS scientists in Milwaukee, Wisconsin in collaboration with Monash University (Selengor, Malaysia) and Sun Yat-Sen University (Guangzhou, China) scientists mapped specific neurons in the brain of tilapia to determine the effects of centrally-administered (precise injection into the brain) ghrelin on these neurons and subsequent effects on blood hormone levels in this fish. Ghrelin treatment affected levels of growth hormone in the pituitary, insulin-like growth factor-I (a growth-promoting hormone) in blood, and growth hormone releasing hormone levels in the hypothalamus area of the brain. These findings demonstrate that ghrelin controls other hormones related to feed intake, growth and immune function via its effects on brain neuropeptides. This knowledge can enhance feeding practices, diet formulations, and selective breeding approaches in ways that improve growth and immune function in commercially-important finfish species.

3. Short-term (dietary) exposure of sexually mature yellow perch to environmentally-relevant methyl mercury (MeHg) levels does not affect reproduction. Commercially produced finfish can be inadvertently exposed to MeHg via environmental and dietary (protein and lipids) sources, but our understanding of the sub-lethal impacts of MeHg on fish reproduction is poor. Understanding the impacts of contaminants, and other stressors, is particularly important to the preservation of genomic resources and crucial to the efficiency and success of breeding and genetic improvement programs. ARS scientists in Milwaukee, Wisconsin in collaboration with University of Wisconsin at Milwaukee, Michigan State University (East Lansing, MI), McGill University (Quebec, Canada), Idaho State University (Pocatello, Idaho) and U.S. Geological Survey (Seattle, WA) scientists found that dietary exposure of yellow perch and zebrafish to MeHg resulted in accumulation of this metal in the tissues of both fish species. Analyses of ovarian gene expression in zebrafish exposed over their entire life-cycle showed changes in several ovarian genes involved in reproductive processes, but there were no changes observed with measures of ovarian structure and function (hormone levels, egg production or embryo mortality). By contrast, ovarian gene expression and measures of ovarian function were not altered by MeHg exposure in yellow perch. These results indicate that environmentally relevant MeHg exposures do not impact the spawning ability of yellow perch within a single season. However, whole life-cycle exposures, similarly to that conducted in zebrafish, should be performed in yellow perch to ensure that environmentally-relevant doses do not affect life-time reproductive capacity of selectively-bred yellow perch broodstocks.

Review Publications
Lee, S., Zhao, H., Li, Y., Binkowski, F., Deng, D., Shepherd, B.S., Hung, S., Bai, S. 2018. Evaluation of formulated feed for juvenile lake sturgeon (Acipenser fulvescens) based on growth performance and nutrient retention. North American Journal of Aquaculture. 80:223-236. https://doi.org/10.1002/naaq.10026
Debofsky, A.R., Klinger, R., Mora, F.X., Waltz, M., Shepherd, B.S., Larson, J., Anderson, D., Yang, L., Goetz, F., Basu, N., Head, J., Tonellato, P., Murphy, C., Carvan, M.J. 2017. Female reproductive impacts of dietary methylmercury in yellow perch (Perca flavescens) and zebrafish (Danio rerio). Chemosphere. 195:301-311. https://doi.org/10.1016/j.chemosphere.2017.12.029.
Shepherd, B.S., Spear, A., Philip, A.M., Leaman, D.W., Stepien, C.A., Sepulveda-Villet, O.J., Palmquist, D.E., Vijayan, M.M. 2018. Effects of cortisol and lipopolysaccharide on expression of select growth-, stress- and immune-related genes in rainbow trout liver. Fish and Shellfish Immunology. 74:410-418. doi: 10.1016/j.fsi.2018.01.003.
Ke, Q., Weaver, W., Pore, A., Gorgoglione, B., Wildschutte, J., Xiao, P., Shepherd, B.S., Spear, A., Malathi, K., Stepien, C.A., Vakharia, V.N., Leaman, D.W. 2017. Role of viral hemorrhagic septicemia virus (VHSV) matrix (M) protein in suppressing host transcription. Journal of Virology. 91(19):e00279-17. https://doi.org/10.1128/JVI.00279-17.