Location: Harry K. Dupree Stuttgart National Aquaculture Research Cntr
2022 Annual Report
Objectives
1. Improve the performance of hybrid striped bass (HSB) fingerlings in intensive production systems.
1.A. Develop fish stocking and dissolved oxygen and dissolved inorganic nitrogen management strategies for intensive production of HSB.
1.B. Optimize production of advanced HSB fingerlings in the biofloc technology production system.
1.C. Determine the nutritional value of alternative ingredient mixes and supplemental exogenous enzymes for hybrid striped bass.
1.D. Evaluate practical fishmeal-free (FMF) and plant-based diets for HSB performance and nutrient (N & P) retention in intensive production systems.
1.E. Determine optimum feeding regime, live feed enrichment, and feasibility of microparticulate diets to increase growth, survival, condition, and fatty acid composition in moronid larvae.
2. Reduce on-farm mortalities to pathogens in HSB aquaculture.
2.A. Perform controlled challenges with white bass, striped bass, palmetto and sunshine hybrid striped bass to establish their susceptibility to emerging pathogens.
2.B. Detect genetic variation for resistance to disease in host.
2.C. Identification of Moronid Dermal Mucus Antimicrobial Peptides and Proteins.
2.D. Development of Natural Antimicrobials for Prophylaxis and Treatment of Bacterial Infections.
2.E. Develop methods for harmful algal bloom control.
3. Develop and implement technologies that enhance genetic improvement of HSB.
3.A. Create and release a first-generation white bass genome and transcriptome assembly.
3.B. Optimize a photo thermal manipulation protocol for offseason (fall) spawning of WB and create even/odd year class base populations for selective improvement of important production traits.
3.C. Identify phenotypic differences in WB for growth and utilization of plant protein- and plant oil-based diets and determine the genetic variation in protein, lipid and carbohydrate utilization for the identified traits.
3.D. Develop methods for the production of triploid HSB.
Approach
Develop fish stocking and dissolved oxygen and dissolved inorganic nitrogen management strategies for intensive production of hybrid striped bass utilizing both traditional and split-pond production systems; Optimize production of advanced hybrid striped bass fingerlings in biofloc technology production system; Determine the nutritional value of alternative ingredient mixes and supplemental exogenous enzymes for hybrid striped bass. Evaluate practical fishmeal-free and plant-based diets for hybrid striped bass performance and nutrient (Nitrogen and Phosphorus) retention in intensive production systems; Determine optimum feeding regime, live feed enrichment, and feasibility of microparticulate diets to increase growth, survival, condition, and fatty acid composition in moronid larvae; Determine optimum larval feeding regime for larval Morone using live feeds to increase growth, survival, and larval quality through metamorphosis; Determine the influence of live feed enrichment on growth, survival, condition, and fatty acid composition of Morone larvae; Determine the feasibility of replacing live feeds with formulated microparticulate diets from first feeding in moronid larvae; Perform controlled challenges with white bass, striped bass, palmetto and sunshine hybrid striped bass to establish their susceptibility to emerging pathogens; Detect genetic variation for resistance to disease in moronids. Identify microbial genes and pathways critical for pathogenesis in moronids; Identification of Moronid dermal mucus antimicrobial peptides and proteins; Development of natural antimicrobials for prophylaxis and treatment of bacterial infections; Develop methods for harmful algal bloom control; Create and release a first-generation white bass genome and transcriptome assembly; Optimize a photo thermal manipulation protocol for offseason (fall) spawning of white bass and create even/odd year class base populations for selective improvement of important production traits; Identify phenotypic differences in white bass for growth and utilization of plant protein- and plant oil-based diets and determine the genetic variation in protein, lipid and carbohydrate utilization for the identified traits; Develop methods for the production of triploid hybrid striped bass.
Progress Report
An intensive study was completed to compare production, for the first time, of market-size hybrid striped bass in conventional earthen ponds to that in earthen ponds modified to the split-pond configuration. Results showed that hybrid striped bass can be grown successfully in split ponds and that in split ponds feed utilization is improved and mean total ammonia-nitrogen concentration is lower. Further evaluation of HSB production in split ponds compared to conventional ponds is underway. Subobjective 1A
A study was completed in outdoor biofloc tanks to evaluate the effect of stocking from 100 to 300 1-2-g striped bass fingerlings per square meter of tank water surface on fingerling growth and yield. Data analysis is on-going. Subobjective 1B
We tested an acid insoluble ash method of estimating nutrient digestibility in hybrid striped bass and rainbow trout diets with 3 levels of ash, 2 indigestible markers and 3 proteins: menhaden fish meal, dried distillers grain with solubles (DDGS), and soybean meal. Diet and fecal analyses are in progress. To determine nutritional value of supplemental feed enzymes on hybrid striped bass and rainbow trout, test ingredients were identified, gathered, and analyzed after supply side shortages abated. Diets will be extruded Aug 2022. Fish were stocked in test tanks July 2022 anticipating feed trials. Digestibility and growth trials will be performed concomitantly. Fish trials will occur Aug–Oct 2022. Subobjective 1C
Due to loss of restaurant sales in the pandemic, hybrid striped bass (HSB) farmers began holding and producing larger fish aimed at retail markets; however, producers claimed current diets result in excessive body fat in these larger fish, potentially putting off retail buyers and consumers. In response to industry requests, we began testing alternative HSB finishing diets in ponds and biofloc grow-out systems. Fiscal Year (FY) 2021 studies evaluated a commercial finishing diet favored by some HSB farmers; however, fish fed this diet had higher fat content at harvest then those fed the current commercial feed. Results spurred a second industry sponsored pond study (in progress FY 2022) to evaluate the effect of ARS formulated finishing diets on HSB yield and composition. Subobjective 1D
We continue to research hybrid striped bass larval weaning to microdiets (MDs) to establish the earliest time larvae can be converted to a MD. Studies this year compared growth of larvae on six MDs to live feed using cofeeding (live feed + MD together), and investigated hybrid striped bass larval gut enzyme development and histology, with our colleagues at University of Arkansas, Pine Bluff, to determine when substitution of a MD is advisable and reveal potential intestinal inflammation caused by MDs. Data analyses are ongoing. Subobjective 1E
We previously determined that white bass (WB) and striped bass (SB), the parental species to hybrid striped bass (HSB), display large differences in susceptibility to the bacterium Flavobacterium columnare. We created F2 backcross HSB by mating F1 HSB with parental WB to be used as a mapping population for disease resistance Quantitative Trait Loci (QTL) studies. Disease challenges on families and sampling has been performed and building of a high-density Moronid single nucleotide polymorphism (SNP) array is underway through pending new Cooperative Agreement. This research will aid in the development of disease-resistance genetic markers. Subobjective 2B
We have developed collaborations with the University of Maryland (UM), Moderna, and the Human Microbiology Institute (HMI) over the past year. Our UM collaboration has been highly successful, and we have identified and established the potency of three endolysins (antimicrobial proteins) against Streptococcus iniae bacteria strains. In our in vitro assays, these endolysins have demonstrated a potency level beyond any known antibiotic that is effective against S. iniae. We are continuing into the next phase whereby we will test the in vivo efficacy of the endolysins in Moronid species. Our Moderna collaboration is in the early phases. Most recently we have signed confidentiality agreement with the HMI, which has developed several new antimicrobial drugs which we plan to test against fish pathogens. Subobjective 2C, 2D
We continue our work on the dermal mucus antimicrobial proteins and have been highly successful in this endeavor. We have developed techniques that led to a 75% increase in the number of antimicrobial proteins in the dermal mucus. These results are very promising and may lead to simple, low-cost methods of disease control. Currently, we are purifying each antimicrobial protein for in vitro testing while simultaneously testing the ability of the proteins to protect Moronid fish species using in vivo testing protocols. Subobjective 2C, 2D
In collaboration with ARS researchers at Aquatic Animal Health Research Unit, Auburn, Alabama and University of Arkansas, Pine Bluff we identified over 20 antimicrobial protein genes towards Streptococcus iniae. Work is ongoing to recombinantly express these proteins and test them as potential treatments for this fish pathogen. Subobjective 2D
We began a toxic algae monitoring program with the Fish Disease Diagnostic Laboratory, University of Arkansas, Pine Bluff, to enumerate and ID algae from ponds. We have identified multiple algal species present in farm ponds, including the toxic algae Aphanocapsa. Suitable treatments are being recommended when algae reach critical thresholds based on our previous research. Subobjective 2E
We continue to breed and selectively improve growth traits in white bass fed a plant-based diet and provide these stocks to stakeholders through Cooperative Agreements. This year, 74 white bass crosses were made, 26,000 fish transferred to grow-out, and the top third were transitioned to the plant-based diet selection diet. This represents significant progress toward advancing genetic improvement of hybrid striped bass by building white bass resources for commercially important traits. Subobjective 3B, 3C
Subsets of the white bass (WB) families created in crosses produced in Subobjective 3.A (15 crosses) were tagged and raised in a common garden tank experiment where they were fed either a fish meal control or all-plant protein diet. The families chosen represented a portion of the progeny derived from five wild and domestic strains of WB. At the end of the feeding trial, the top ~ 10% and bottom ~ 5% performing crosses were selected for whole body composition and blood collection for gene expression analyses (RNAseq) based on the high-quality WB genome and transcriptome generated in Subobjective 3.A. Analyses were completed this year and a peer-reviewed manuscript is in development. Subobjective 3C
We continue to collaborate with researchers at Keo Fish Farm, Keo, Arkansas, and University of Arkansas, Pine Bluff to produce sterile triploid hybrid striped bass. We previously developed methods to reduce hybrid striped bass egg adhesion during incubation because standard industry methods interfered with triploid production; Keo continues to use these methods exclusively. Our previous attempts at using pressure to make triploid fish yielded marginal results; methods will be modified for next spawning season. Timing of 2nd polar body release continues. Results were limited by the COVID pandemic, water use needs at the commercial hatchery and flow-cytometry analyses difficulties. A University lab was contracted to analyze flow-cytometry samples for ploidy next year. Subobjective 3D
We continue our collaborations with researchers at the Freshwater Institute (Shepherdstown, West Virginia) and Hood College (Frederick, Maryland) on the toxicity of peracetic acid to early life stages of Atlantic salmon. This research is also investigating the use of peracetic acid in recirculating aquaculture systems to eliminate pathogens.
Our collaboration with researchers in Denmark, Germany, Norway and the U.S. has continued to establish the importance of peracetic acid as a disinfectant to the global aquaculture industry. In the U.S., the major manufacturer of peracetic acid (PeroxyChem) currently has an EPA Registration to use their products as an aquaculture disinfectant and are considering other possible uses for their products.
We continue to examine the effects of water source and diet on microbiome diversity in Nile Tilapia. Microbial and fungal communities are being examined in gut contents and water samples after grow out experiments comparing earthen ponds to biofloc systems. High-throughput sequencing is being used to characterize diversity and variation in all samples.
In collaboration with ARS researchers at Aquatic Animal Health Research Unit in Auburn, Alabama, we continue to examine the genetic effects of acute hepatopancreatic necrosis disease (AHPND) in pacific white shrimp. Proteins found in the bacteria Vibrio parahaemolyticus have been shown to be major inducers of AHPND, an ongoing problem in the shrimp farming industry. Shrimp were challenged with recombinant Vibrio induction proteins and time-course tissues were sampled. RNA sequencing results between challenged and control groups are currently being examined.
In collaboration with ARS researchers at Hagerman Fish Culture Experiment Station in Hagerman, Idaho, the University of Idaho, and Auburn University, we continue to analyze large multi-tissue RNA sequencing datasets of rainbow trout to determine genetic differences in innate immune responses to the fish pathogen Flavobacterium psychrophilum. Several strains of rainbow trout are being examined that have shown varying degrees of susceptibility to this pathogen.
Accomplishments
1. Organic acids and essential oils improve growth rates, feed efficiency, and disease resistance in carnivorous fish. ARS researchers in Stuttgart, Arkansas, with ARS colleagues at the Small Grains and Potato Germplasm Research Center, Hagerman, Idaho, and researchers at the US Fish and Wildlife Service Bozeman Fish Technology Center, Montana performed feeding trials with hybrid striped bass and rainbow trout to test the efficacy of organic acids and essential oils from herbs and spices as alternatives to antibiotics to combat Streptococcus infection in hybrid striped bass and cold-water disease in rainbow trout. Results showed that both organic acids and essential oils improves growth rate and feed efficiency in rainbow trout and hybrid striped bass. The results from phase two studies (disease challenges, gene regulation analyses, and hybrid striped bass taste test panels) convinced a major aquafeed supplier to market a separate line of feed containing essential oils.
2. Striped bass are highly susceptible to common aquaculture diseases – possible target for selection. ARS researchers previously demonstrated the susceptibility of multiple aquaculture species to several common diseases, but the need to establish the susceptibility of hybrid striped bass (HSB) as well as their parental species (white bass WB and striped bass SB) to the most common aquaculture diseases plaguing the HSB industry was recently identified by producers as an area of high priority. To address this pressing industry need, ARS researchers in Stuttgart, Arkansas, conducted a series of disease challenge studies in these species to the three most common aquaculture diseases identified by the HSB industry: columnaris disease, motile aeromonad septicemia and streptococcosis. Following challenge, only 1% of SB survived across the three diseases and died twice as early as WB and HSB. These results established that WB are the most resistant to all three diseases, SB are most susceptible, and HSB are intermediate to the two parental species. We are currently working with our North Carolina State University partners to incorporate these results into the ARS – NCSU HSB Selective Breeding Program to incorporate SB disease resistance as a selection trait for potential improvement of HSB disease resistance.
3. The palmetto bass is almost completely resistant to common aquaculture diseases while the foodfish standard sunshine bass is highly susceptible. Sunshine bass (SSB), a hybrid cross using a female white bass (WB) and a male striped bass (SB), is the dominant fish used in the hybrid striped bass (HSB) foodfish industry while the palmetto bass (PB), another HSB cross, uses a female SB and a male WB; these two hybrids possess different production characteristics. Previous research in our lab demonstrated the disease tolerance in HSB parental species relative to the SSB, however nothing was known about the disease tolerance of PB. ARS researchers in Stuttgart, Arkansas, conducted a series of foundational experiments to investigate the susceptibility of these hybrid species to two common pathogens plaguing the HBS industry, columnaris disease and motile aeromonad septicemia. Results demonstrated that the hybrids were significantly different in their survival to both diseases, with PB showing near complete resistance to both diseases (> 95% survival), whereas SSB was highly affected by both diseases (< 5% survival), and that susceptibility to disease for each hybrid appears to mimic the pattern found in their paternal parental species. These studies will aid farmers and researchers with management strategies to combat infectious diseases, aid in the development of new therapeutants, and provide HSB producers additional species for production when faced with diseases in their production environments.
4. Zinc: the cheaper, safer alternative to copper for preventative pathogen treatments in aquaculture. Copper has been used as a water treatment for prophylaxis against bacterial infections for decades; however, the price of copper has risen to $10K per ton while the demand has increased. Zinc is a closely related element yet has a market price of $3K per ton. ARS researchers in Stuttgart, Arkansas, explored the possibility of zinc as an alternative to copper as a water decontaminant using a series of toxicity trials with largemouth bass fry. These results revealed that zinc is just as effective as copper in its ability to kill fish pathogens yet is only half as toxic to fish, providing farmers a cheaper, safer treatment for the prevention of disease outbreaks in aquaculture.
5. Finishing diet makes fish fattier. The COVID-19 pandemic disrupted traditional marketing channels for many hybrid striped bass (HSB) farmers, causing them to seek alternative markets for their fish. With much of the domestic HSB crop typically sold either live or as whole fish on ice, these potential new market opportunities for fish larger than 1.5 lbs have farmers concerned that larger fish could have excessive fat content, causing consumer dissatisfaction. In a HSB production experiment, ARS scientists in Stuttgart, Arkansas, found that fish fed a commercial finishing diet used by some farmers to produce leaner, larger market-size fish actually resulted in fattier fish when compared to a standard production diet. This information has been communicated to HSB farmers for use in making management decisions and improving product quality as they develop new markets when producing these larger fish.
6. Marine fish meal can be replaced by alternative proteins in bass diets. Marine fish meal (FM) is the most nutritious, and most expensive, ingredient in many fish diets, but is a limited resource due to static populations of the fish species used in its production. Thus, replacing FM is essential if aquaculture diets are to be sustainable. ARS researchers in Stuttgart, Arkansas, along with their colleague at Iowa State University, initiated trials comparing diets having varying levels of soybean meal or poultry by-product meal, partially or completely replacing FM, with one test diet consisting exclusively of plant protein sources. All test diets yielded similar growth performance as fish fed a 30% FM control diet apart from an all-plant diet and a commercial protein blended product which resulted in reduced performance. This indicates that WB can be fed diets in which some, if not all, FM can be replaced by more sustainable, and cost-effective, ingredients. Results are currently being used in the ARS – NCSU HSB Selective Breeding Program to select a WB (to produce HSB) with superior performance on alternative (plant-based) feeds.
7. Gene expression was altered in cultured Florida pompano following ectoparasite Amyloodinium infection. The disease-causing ectoparasite Amyloodinium ocellatum is a nuisance in cultured marine and brackish water fishes worldwide, including the highly desirable pompano, with its high economic value in the U.S. This parasite infects fish gills and causes tissue damage, increased respiratory rate, and reduced appetite and mortality, especially in recirculating aquaculture systems. ARS researchers in Stuttgart, Arkansas, along with their colleagues at Auburn University, conducted studies which mimicked a natural infection in cultured pompano where they sought to identify fish genomic responses following infection. Researchers found that genes involved with immune response and inflammation suppression, cellular respiration, tissue repair, and food intake and fat metabolism were all affected. These genes all play an important role in a fish’s ability to fight disease and maintain cellular health. This work provides the first genomic report in pompano which is key to understanding this parasitic disease. This will aid in the design of disease monitoring strategies, future selection for disease resistance in pompano, and the development of new drug treatment measures.
8. ARS strain of rainbow trout selected for tolerance to plant-based diets displays enhanced non-specific immunity to infectious hematopoietic necrosis virus (IHNV). IHNV infections in trout and salmon can result in high levels of mortality and significant economic losses for aquaculture producers. ARS researchers in Stuttgart, Arkansas, in collaboration with ARS researchers at Hagerman Fish Culture Experiment Station in Hagerman, Idaho, the University of Idaho, and Auburn University examined responses to acute IHNV infection on three major strains of rainbow trout (RBT) used in US aquaculture production: IHNV resistant, viral resistant, and an ARS strain selected for utilization of plant-based feeds. Results demonstrated that strains varied in IHNV resistance, innate immunity and gene expression profiles across multiple tissues and multiple time-points as the disease progressed. These findings highlight the disparity in IHNV resistance in RBT produced in the US, offered insight into the mechanisms of RBT disease response, and revealed the connection between fish innate immunity and utilization of plant-based diets.
9. Teaching aquaponics increases students’ interest in science, technology, engineering, and mathematics (STEM) and spurs interest in environmental science. There is a need for secondary schools in the U.S. to get students interested in, and prepared for, jobs that are based in (STEM) as there is a critical shortage and need in these disciplines. However, many students are very reluctant to take STEM courses. Thus, secondary schools need to provide more hands-on, fun ways to teach STEM disciplines and specifically, more project-based investigation (PBI) environments in the classroom to pique student's interests. Aquaponics is a good candidate for hands-on classroom learning since it involves all the STEM disciplines as well as environmental sciences and ecology. ARS researchers in Stuttgart, Arkansas, in collaboration with Kentucky State University investigated how an aquaponics project-based investigation (APBI) unit affected high school students’ attitudes and interest towards STEM, environmental sciences and ecology, as well as aquaculture and aquaponics. These results demonstrated that a hands-on aquaponics curriculum contributed to students’ positive attitudes toward STEM and provided meaningful learning of ecological concepts, aquaculture, and aquaponics. These APBI teaching models may promote student learning of scientific concepts, increase students’ interest in STEM-related disciplines, and should be considered when schools are developing STEM curricula to develop the next generation of scientists in the U.S.
Review Publications
Mccann, K.M., Rawles, S.D., Lochmann, R.T., Mcentire, M.E., Sealey, W.M., Gaylord, T., Webster, C.D. 2021. Dietary replacement of fishmeal with commercial protein blends designed for aquafeeds in hybrid striped bass (Morone chrysops × Morone saxatilis): Digestibility, growth, body composition, and nutrient retention. Aquaculture Reports. 21. Article 100903. https://doi.org/10.1016/j.aqrep.2021.100903.
Liu, D., Pellicer, A.M., Brüggmann, A., Kiggen, M., Behrens, S., Good, C., Straus, D.L., Meinelt, T. 2021. Effect of water hardness/alkalinity and humic substances on the toxicity of peracetic acid to zebrafish embryos and pathogenic isolates. Aquaculture Reports. 21:10090. https://doi.org/10.1016/j.aqrep.2021.100900.
Green, B.W., Ray, C.L. 2022. Evaluation of settling chamber hydraulic retention time in a Sunshine bass biofloc production system. North American Journal of Aquaculture. 84:165-171. https://doi.org/10.1002/naaq.10229.
Buley, R.P., Adams, C., Belfiore, A.P., Fernandez-Figueroa, E.G., Gladfelter, M.F., Garner, B., Straus, D.L., Wilson, A.E. 2021. Field evaluation of seven products to control cyanobacterial blooms in aquaculture. Environmental Science and Pollution Research. 28:29971-29983. https://doi.org/10.1007/s11356-021-15753-x.
Ghosh, S., Straus, D.L., Good, C., Phuntumart, V. 2021. Development and comparison of loop-mediated isothermal amplification with quantitative PCR for the specific detection of Saprolegnia spp. PLoS ONE. 16(12):e0250808. https://doi.org/10.1371/journal.pone.0250808.
Zhang, D., Mohammed, H., Ye, Z., Rhodes, M., Thongda, W., Zhao, H., Jescovitch, L., Fuller, S.A., Davis, A., Peatman, E. 2022. Transcriptomic profiles of Florida pompano (Trachinotus carolinus) gill following infection by the ectoparasite Amyloodinium ocellatum. Fish and Shellfish Immunology. 125:171-179. https://doi.org/10.1016/j.fsi.2022.05.017.
Fuller, S.A. 2022. Determining the optimum feed duration of Artemia nauplii to maximize hybrid striped bass larval growth in cool-water culture conditions. Journal of Applied Aquaculture. 34(2):267-278. https://doi.org/10.1080/10454438.2020.1841059.
Bledsoe, J.W., Ma, J., Cain, K., Bruce, T., Rawles, A.A., Abernathy, J.W., Welker, T.L., Overturf, K.E. 2022. Multi-tissue RNAseq reveals genetic and temporal differences in acute viral (IHN) infection among three selected lines of rainbow trout with varying resistance. Fish and Shellfish Immunology. 124:343-361. https://doi.org/10.1016/j.fsi.2022.03.034.
Lange, M.D., Abernathy, J.W., Farmer, B.D., Beck, B.H. 2021. Use of an immersion adjuvant with a Flavobacterium columnare recombinant protein vaccine in channel catfish. Fish and Shellfish Immunology. 117:136-139. https://doi.org/10.1016/j.fsi.2021.07.025.
Green, B.W. 2022. Fertilizer use in aquaculture. In Davis, D.A., editor. Feed and Feeding Practices in Aquaculture. Second edition. Cambridge, MA: Woodhead Publishing. p. 29-63. https://doi.org/10.1016/B978-0-12-821598-2.00012-6
Roy, L.A., Rawles, S.D., Quintero, H.E., Kelly, A.M., Park, J., Webster, C.D. 2021. Weight loss, survival, and fatty acid composition in over-wintered juvenile coppernose bluegill (Lepomis macrochirus purpurescens) cultured in outdoor tanks using different feeding regime. North American Journal of Aquaculture. 84(1):42-52. https://doi.org/10.1002/naaq.10213.
