Location: Harry K. Dupree Stuttgart National Aquaculture Research Cntr
2020 Annual Report
Objectives
Objective 1. Characterize the cellular and molecular immune responses governing resistance/susceptibility to infectious disease (Flavobacterium columnare), and develop strategies to enhance immune protection through immunomodulatory compounds, nutritional status, or water chemistry.
Subobjective 1A. Describe the cellular, transcriptional, and pathological responses of mucosal tissues in healthy and Flavobacterium columnare-challenged fish.
Subobjective 1B. Investigate the effects of copper sulfate, short-term feed deprivation, and water chemistry on the mucosal immune system.
Objective 2. Assess microbial community diversity, prevalence, and distribution under homeostatic conditions, after columnaris disease challenge, and following environmental perturbation.
Subobjective 2A. Profile the composition and population dynamics of microbes that reside on the skin and gill of healthy and columnaris-challenged fish.
Subobjective 2B. Investigate the effects of copper sulfate, short-term feed deprivation, and water chemistry on microbial community composition.
Approach
In Objective 1 our research team will utilize next-generation sequencing to establish temporal profiles of transcriptional responses to Flavobacterium (F.) columnare in gill and skin; use flow cytometry to monitor the type and abundance of immune cells trafficking to the gill of challenged fish; and characterize the histopathological changes to an F. columnare challenge and draw parallels to the shifts in immune-related transcripts and cell phenotypes. Studies will also examine the transcriptional and immunological consequences to alterations in water chemistry and to chemical compounds such as copper sulfate. In Objective 2 the core microbiota associated with mucosa in healthy fish and F. columnare infected fish will be characterized using high-throughput 16S v4 amplicon sequencing. This work will identify the core microbial communities commonly residing in the mucosal barriers of both warmwater bass and channel catfish; vital information towards identifying bacterial species that can be exploited to improve fish health.
Progress Report
This is the final report for project 6028-32000-007-00D, which has been replaced by the new project 6028-31630-009-00-D, "Enhancing the Production of Hybrid Striped Bass Through Improved Genetics, Nutrition, Production Management, and Fish Health." For additional information, see the new project report.
Over the life of this 5-year project, objectives have been met and exceeded, with outcomes reported to stakeholders through presentations at major international conferences and peer-reviewed publications. Technology transfer has been accomplished as well as cooperative agreements put in place based on the research during this project period. Objective 1 was to characterize the cellular and molecular immune responses governing resistance/susceptibility to infectious disease (Flavobacterium columnare) and develop strategies to enhance immune protection through immunomodulatory compounds, nutritional status, or water chemistry. Subobjectives included describing responses of mucosal tissues in healthy and Flavobacterium columnare-challenged fish and investigating copper sulfate effects on the fish mucosal immune system. In this regard, ARS researchers developed a host-pathogen research model using both channel catfish and hybrid striped bass with the pathogen Flavobacterium columnare. Using this model in catfish has led to the discovery of Flavobacterium DnaK proteins as critical in host mucosal immunity, for which a vaccine candidate is in trials. Using copper sulfate as a treatment for Flavobacterium columnare infection revealed that host gene expression responses are critical to catfish protection and that alternative gene-splicing events can occur between treatment and control mucosal tissues. For the hybrid stiped bass model, variation between parent species - white bass and striped bass - and the hybrid offspring during Flavobacterium columnare infection have been revealed, for which a genetic mapping model for disease resistance to columnaris disease is being built. The major focus of Objective 2 was to assess Flavobacterium columnare during or after some stimuli. Therefore, much research under this objective was performed on Flavobacterium columnare transition from free-living planktonic cells through the formation of biofilm during fish infection. From this research, ARS researchers identified that iron-acquisition systems are critical for Flavobacterium columnare biofilm formation and, therefore, critical in its pathogenicity. Existing and novel compounds are currently being evaluated using this system as a potential target for advanced therapeutants.
ARS researchers have previously determined that sunshine hybrid striped bass, and its maternal parent white bass, display differences in their susceptibility to the bacterium, Flavobacterium columnare, whereby hybrid striped bass incurs significantly higher mortality when challenged under laboratory conditions compared to white bass. Studies to evaluate the transcriptome through RNA sequencing of white bass and hybrid striped bass gill tissue after Flavobacterium columnare challenge are currently being conducted. This research will provide a better understanding of the host-pathogen interactions that are important in the production of Morone spp. in order to improve treatments and aid in the development of disease-resistance genetic markers.
ARS researchers have determined that white bass, striped bass, and its hybrid display differences in susceptibility to additional pathogens important throughout aquaculture including Aeromonas hydrophila. Research is ongoing to evaluate disease susceptibility in gram positive bacteria including Streptococcus species. Characterizing variation in disease resistance traits will ultimately aid in the development of disease-resistance genetic markers and production of effective therapies.
ARS researchers in Stuttgart, Arkansas, and Auburn, Alabama, continue to evaluate vibrio toxin effect on shrimp using RNA sequencing of hemolymph tissues. By studying this important system in shrimp, researchers hope to elucidate molecular mechanisms that make shrimp susceptible to pathogens.
ARS researchers continue to examine mucus formation and the interplay with disease susceptibility in additional fish species including white bass, striped bass and hybrid striped bass. The interaction of moronid species mucus with additional pathogens that affect the hybrid striped bass industry, including Aeromonas and Streptococcus species, are currently under investigation. These studies will aid in our understanding of variation in disease resistance traits in moronids and aid in the development of novel therapeutants.
ARS researchers are in the beginning stages of examining the antimicrobial properties of white bass mucus, which is the most disease resistant species of the moronid basses to Flavobacterium columnare. These studies will aid in our understanding of variation in disease resistance traits in moronids and aid in the development of novel therapeutants.
ARS researchers continue to investigate ambient water temperature effects during the initiation of a mucosal adaptive immune response in the skin of multiple fish species. This work examines the humoral immune response under various environmental temperature conditions including those represented in industry to identify the ideal time and conditions for immunizing finfish.
ARS researchers initiated a collaboration with researchers at the Freshwater Institute, Shepherdstown, West Virginia, on the toxicity of peracetic acid to early life stages of Atlantic salmon. This collaboration is also investigating the use of peracetic acid in recirculating aquaculture systems to treat these systems with peracetic acid to eliminate pathogens.
ARS researchers continued a collaboration with researchers at the University of Arkansas at Pine Bluff, Pine Bluff, Arkansas and Keo Fish Farm, Keo, Arkansas to produce triploid (sterile) hybrid striped bass. In a 2-year-old cohort produced via heatshock on eggs, we determined growth rates and morphology of these triploid fish as compared to diploid (normal) fish.
ARS researchers continued collaborating with researchers in Denmark, Germany and the US, to establish the importance of the potent disinfectant peracetic acid to the global aquaculture industry. After evaluating our research, the major manufacturer of peracetic acid in the U.S. (PeroxyChem) and another company
that retails fishery products (AquaTactics) have gained EPA Registrations to use their products in the aquaculture industry as a disinfectant.
Accomplishments
1. Fish mucus stimulates Flavobacterium columnare biofilm formation and the upregulation of iron acquisition system pathways. Flavobacterium columnare is the bacteria responsible for columnaris disease, an important fish pathogen that causes significant losses to warmwater fish production. ARS researchers in Stuttgart, Arkansas, in collaboration with an ARS researcher in Auburn, Alabama, characterized factors that stimulate the transition of planktonic, or free-living, Flavobacterium columnare to the formation of biofilms. Biofilms are a critical stage of bacterial pathogenesis which requires a sustained host-pathogen interaction at mucosal sites including the fish gill and skin. ARS researchers identified that skin mucus from different fish species can stimulate the formation of biofilms to varying degrees. They further found that iron acquisition systems are upregulated in biofilm cells during their transition from being planktonic cells. Understanding biofilm formation and the molecular mechanisms behind this action will benefit the development of new vaccines and novel therapies to treat infectious diseases in aquaculture.
2. Disease resistance of white bass, striped bass and hybrid striped bass to Flavobacterium columnare and the mucosal effects on the causative bacteria. Columnaris disease generates substantial losses of many freshwater fish species including hybrid striped bass, a major aquaculture species. ARS researchers in Stuttgart, Arkansas, and Auburn, Alabama, determined the susceptibility of white bass, striped bass, and hybrid striped bass to columnaris disease and investigated the effects of bass mucus on total bacterial growth and biofilm formation. ARS researchers confirmed that white bass are more resistant to columnaris disease than hybrid striped bass and that striped bass were the most susceptible. Results also suggested that one component of variation in moronid disease resistance may be contained in the fish mucus and that survival of resistant fish is likely associated with innate factors such as antimicrobial effectors present in the mucus. Results will be of use in further elucidation of mechanisms behind disease susceptibility in moronid bass species for therapeutant development and in identifying genetic markers useful in the selection of parental species for the production of improved hybrid striped bass.
Review Publications
Good, C., Davidson, J., Straus, D.L., Harper, S.B., Marancik, D., Welch, T.J., Peterson, B.C., Pedersen, L., Lepine, C., Redman, N., Meinelt, T., Liu, D., Summerfelt, S. 2020. Assessing peracetic acid for controlling post-vaccination Saprolegnia spp.-associated mortality in juvenile Atlantic salmon Salmo salar in freshwater recirculation aquaculture systems. Aquaculture Research. 00:1-4. https://doi.org/10.1111/are.14567.
Lange, M.D., Farmer, B.D., Abernathy, J.W. 2019. Vertebrate mucus stimulates biofilm development and upregulates iron acquisition genes in Flavobacterium columnare. Journal of Fish Diseases. https://doi.org/10.1111/jfd.13103.
Liu, D., Lazado, C.C., Pedersen, L., Straus, D.L., Meinelt, T. 2020. Exogenous reactive oxygen species alter antioxidative state and immunological responses of rainbow trout. Fish and Shellfish Immunology. https://doi.org/10.1016/j.aquaculture.2020.734956.
