Research Project: Developing Nutritional, Genetic, and Management Strategies to Enhance Warmwater Finfish Production

Location: Harry K. Dupree Stuttgart National Aquaculture Research Cntr

2019 Annual Report

Objectives
Objective 1: Develop novel ingredients and formulate diets for warmwater finfish. Novel ingredients and new formulations will target reduced reliance on fishmeal in the diets, enhanced nutrient delivery (e.g., better amino acid availability), and reduced anti-nutritional factors. Sub-objective 1A: Develop and validate practical all-plant protein diets for hybrid striped bass (HSB) and white bass (WB). Sub-objective 1B: Determine optimal inclusion level of distiller’s dried grains with solubles and high-protein distillers dried grains with solubles in diets for Nile tilapia. Sub-objective 1C: Determine optimal inclusion level of cottonseed meal and poultry by-product meal in diets for largemouth bass. Objective 2: Obtain and test white bass broodfish from multiple populations for improved production of hybrid striped bass. Key phenotypic traits will be defined and a white bass broodstock will be established. Sub-objective 2A: Obtain and produce white bass from multiple geographically distinct populations. Sub-objective 2B: Conduct white bass strain performance evaluation and selection. Sub-objective 2C: Hybrid striped bass performance evaluation. Objective 3: Compare and optimize production of hybrid striped bass in biofloc technology and pond production systems. Sub-objective 3A: Define stocking rate/initial biomass – yield relationship for HSB in a biofloc technology system. Sub-objective 3B: Compare production of HSB in biofloc technology and pond production systems.

Approach
Identify promising feed palatants that enhance all plant protein (APP) diet feed intake; identify plant proteins for use in APP test diets; formulate a series of marine fish meal (FM) replacement diets using ideal protein theory, plant protein blends, and feed additives designed to ameliorate ingredient deficiencies; characterize growth performance, nutrient retention, gut microflora, and metabolic gene expression in response to FM replacement diet; and validate promising APP diets in a pond production trial. Feeding studies will evaluate the effect of partial or total replacement of FM by either Distiller’s dried grains with solubles or high protein Distiller’s dried grains with solubles in the diet of Nile tilapia on feed intake, growth, feed conversion, and histology of the intestine. A feeding study will evaluate the effect of substitution of FM by various ratios of poultry by-product meal and cottonseed meal in practical diets for juvenile largemouth bass on growth performance, health status, and body composition. Adult white bass (WB) will be acquired from three distinct regions of the species’ native range (Arkansas River, AR, Central and South TX, and Upper Tennessee River, TN), spawned and the genetic diversity and production performance of multiple strains and families will be compared in tanks and ponds to our domesticated WB broodfish population in order to select superior performing families to use in developing improved synthetic lines. Evaluate production performance of families of hybrid striped bass (HSB) made from the WB improved synthetic lines. Quantify the relationship between the HSB stocking rate/initial biomass – yield relationship to produce advanced fingerlings in the biofloc technology production system and compare to traditional pond production method.

Progress Report
ARS scientists in Stuttgart, Arkansas, continued a pond study (season 2) to compare market-sized hybrid striped bass (HSB) performance when fed either all-plant protein diets, fish meal-free diets, a fish meal control diet, or a commercial feed. Fish were stocked as juveniles in Spring 2018 and grown to one-pound average size, i.e., advanced food-fish stockers, over the course of a year using a commercial feed common to the industry. In Spring 2019, fish in all the ponds were inventoried for starting weights and numbers and feeding of the test diets was initiated. Harvest of 2.5-3.5 lb market-weight fish is anticipated in fall 2019. In addition to dietary protein composition, the diets also test whether a commercial form of the enzyme phytase can release plant-bound phosphorus (P) and eliminate adding P to the diet, thus reducing pollution. This study is the culmination of several studies to optimize fish meal replacement in hybrid striped bass diets by vetting diets in pond production. (Objective 1) Phosphorus is present in plant feedstuffs as phytate, which is not available to fish because they cannot digest it. Phytase is an enzyme that releases the phosphorus from the phytate. Reduction of phytate as an anti-nutrient, particularly in the context of improvement of health parameters and skeletal mineralization, has not yet been considered in catfish when fed plant protein-laden aquafeeds. ARS scientists in Stuttgart, Arkansas, conducted a feeding study in nylon floating cages to determine if amending a commercially-available catfish diet with different doses of a second-generation phytase affects growth, body composition, hematological parameters, and immune function in channel catfish. After 120 days, the study was concluded, and fish were measured for growth, while some fish were randomly sampled for body composition and analysis. Liver samples were also obtained from several fish for genomic analysis. All data, except liver genomic analysis, have been collected and will be statistically analyzed when the genomic analysis is completed. (Objective 1) ARS scientists in Stuttgart, Arkansas, have been seeking to determine the earliest time that HSB larvae can be converted to a prepared diet by minimizing the time spent on live food items all while maximizing larval growth and survival. HSB larvae typically spend the first portion of exogenous feeding in tank production consuming rotifers (Brachionus sp.) before moving on to larger Artemia nauplii, and finally weaning onto manufactured diets. The current study focused on developing feeding protocols that minimize the number of days larvae are fed Artemia nauplii. Survival and growth data are currently being analyzed to determine the feeding regime that best maximizes larval growth and survival. This is crucial as the longer larvae are on live feeds, the greater the risks of catastrophic loss and the higher the costs to fish farmers involved in HSB tank production. (Objective 2) The use of selective breeding to produce larger larvae at hatch could provide several advantages for HSB production. Larger larvae might be weaned from live feeds more quickly, have generally higher survival rates, and show improved growth and survival. The goal of the current study, performed by ARS scientists in Stuttgart, Arkansas, was to determine the parental effects of wild and domesticated white bass on the size of larvae. Data analysis is ongoing, and results will be used to inform the current white bass broodstock development program. (Objective 2) White bass (WB) is one of the parental species of hybrid striped bass, a major U.S. aquaculture species. Genetic markers are critical for evaluating population diversity, detecting inbreeding, differentiating unknown individuals by population origin, and, ultimately, selecting for performance traits. Additionally, this may help alleviate the need for wild caught WB in hatchery operations. ARS researchers in Stuttgart, Arkansas, in collaboration with scientists from Auburn University developed genetic markers through genotyping-by-sequencing that could differentiate wild stocks from the only existing domestic stock of WB. Genotyping revealed enough genetic diversity existed to establish a new WB line and ARS scientists in Stuttgart, Arkansas, established a base population using a full-factorial cross in which males and females from each population were bred with males and females from all other populations. All available wild stocks of WB collected from distinct geographical locations, including from waterways in Arkansas, Texas, Alabama and North Carolina, and the domesticated line were utilized in creation of an even year-class base population. A total of 98 families were spawned, maximizing genetic diversity and limit inbreeding, which is also to capacity of current facilities. Rearing of these families to sexual maturity is on-going. (Objective 2) ARS scientists in Stuttgart, Arkansas, began a 6-month feeding trial to select the current year-class of WB based on a fish meal-free diet specially formulated with feed industry collaboration. Fish meal in a typical commercial style formula was replaced with a combination of soybean meal and poultry by-product meal and supplemented with limiting amino acids based on the latest feed formulation theory. Fish are being monitored monthly for feed intake, weight gain, and feed efficiency and will be subjected to a final cull at the end of the summer. At that time top performers from each family will be individually tagged and transferred to common garden rearing tanks in order to produce spawning adults for the FY 20-21 year class. (Objectives 1 and 2) White bass naturally reproduce in the springtime, with the help of natural cues including daylight and temperature. ARS researchers in Stuttgart, Arkansas, developed and implemented a method to further spawn white bass in the offseason (fall) to evaluate additional performance traits, including disease-resistance and tolerance to diet reformulations. Conditioning white bass for fall spawning is ongoing. After one successful year-class spawn utilizing these protocols efforts to improve reproductive success for subsequent year-classes are ongoing. (Objective 2) ARS researchers in Stuttgart, Arkansas, continue to make progress toward a better understanding of the moronid sex-determination system, which currently is uncharacterized. Research continues that examines whether thermal protocols can influence sex ratios in moronids. Sex markers are currently in development and a total of 150 male and 150 female white bass genomes have been re-sequenced and genetic mapping between the populations is underway. (Objective 2) ARS researchers in Stuttgart, Arkansas, in collaboration with researchers at North Carolina State University created hybrid striped bass utilizing highly inbred parent white bass and striped bass. These HSB will be used to create backcross progenies from families that display extremes in important production traits for genome-wide association studies. HSB and their parentals currently are being conditioned for F2 segregate analyses. Efforts to create additional mapping populations are ongoing. (Objective 2) Establishment of additional domesticated broodstocks ready for trait selection is a highly desirable step toward hybrid striped bass improvement. Commercially important traits in hybrid striped bass are heritable, and a single generation of domestication heritably changes gene expression in fish. Thus, ARS researchers in Stuttgart, Arkansas, are investigating the use of HSB in expression quantitative trait loci (eQTL) studies. First-generation HSBs from reciprocal matings of wild and domestic white bass and striped bass were created. A total of 36 fish (original and reciprocal matings) transcriptomes have been sequenced and signatures of domestication currently are being analyzed to distinguish parent-of-origin bias from lineage-of-origin (potential eQTLs for domestication) bias. (Objective 2) Significant progress was made by ARS scientists in Stuttgart, Arkansas, and researchers at North Carolina State University to assemble and improve the white bass and striped bass genome sequences. (Objective 2) ARS scientists in Stuttgart, Arkansas, made significant progress on a study as part of an 1890 Capacity Building Grant to Kentucky State University to evaluate the effect of flow rate (retention time) on denitrification in external solids removal-denitrification tanks coupled with hybrid striped bass biofloc technology production tanks. Three flow rates each are being evaluated in triplicate tanks. Water quality variables are analyzed weekly and fish production data will be evaluated at harvest, which will occur in October. (Objective 3)

Accomplishments
1. Commercial protein blends for hybrid striped bass (HSB) diets. In subordinate project 6028-31630-008-02T “Minimizing Fishmeal in Hybrid Striped Bass Diets Using Commercial Protein Concentrates for Use in Aquafeeds”, ARS scientists at Harry K. Dupree Stuttgart National Aquaculture Research Center, Stuttgart, Arkansas, replaced fish meal with each of four commercially available protein blends (Elite 60™, Elite 65™, Elite 70™, Procision™) produced by HJ Baker and Sons and supplemented limiting nutrients based on the latest diet formulation theory. Test diets were extruded by our cooperator at U.S. Fish & Wildlife Service Bozeman Feed Technology Lab to mimic commercial practices and fed to juvenile hybrid striped bass in tanks for 13 weeks. Good growth, composition of growth, and lack of major differences in innate immunity and hematological parameters were found in fish fed test diets containing any of the three Elite™ fish meal replacers. Considering the high cost ($2,700/ton), lower feed intake, and lower growth performance of the Procision™ test diet, this product does not appear to be a suitable alternative to fish meal in HSB diets. This accomplishment provides science based information to feed and fish producers on cost-effective and sustainable alternatives to fish meal in hybrid striped diets.

2. Optimum hybrid striped bass (HSB) larval feeding regime using rotifers determined. Scientists at Harry K. Dupree Stuttgart National Aquaculture Research Center, Stuttgart, Arkansas, have been seeking to determine the earliest time that Morone larvae can be converted to a prepared diet by minimizing the time spent on live food items all while maximizing larval growth and survival. In the current study, HSB larvae (4 dph; 3.72±0.13mm SL) were stocked in replicated 110-L recirculating tanks at 75/L, maintained at one of two rearing temperatures (18°C, 26°C) and fed rotifers (Brachionus sp.) at increasing two day increments from two days up to twelve days at a set ration of 60 rotifers/mL/d. Following the prescribed period of rotifer feeding, larvae were switched to microcyst Artemia nauplii at a rate of 12 nauplii/mL/d until the end of the study period. Lower rearing temperatures yielded higher survival across all feeding treatments as compared to the higher temperature treatment (p < 0.0001;18°C mean across all treatments 51.8±4.5%; 26°C mean across all treatments 30.2±0.9%). After the 12-day rearing period, growth was maximized by feeding 2-4 days of rotifers at 26°C (p < 0.0001; 10.97±0.84mm TL). Maximal larval growth at 18°C occurred after 2 days of rotifer feeding (8.44±0.84mm TL), however there was no significant difference amongst any of the treatments (p > 0.05). By reducing the time live feeds are needed, the risks and costs to fish farmers involved in hybrid striped bass production are greatly reduced.

Review Publications
Lange, M.D., Abernathy, J.W., Farmer, B.D. 2019. Evaluation of Flavobacterium columnare DnaK recombinant protein vaccine as a means of protection against columnaris disease in channel catfish, Ictalurus punctatus. Frontiers in Immunology. p. 1-13. https://doi.org/10.3389/fimmu.2019.01175.
Lange, M.D., Abernathy, J.W., Farmer, B.D. 2018. Catfish mucus alters the Flavobacterium columnare transcriptome. FEMS Microbiology Letters. 365(22)1-13. https://doi.org/10.1093/femsle/fny244.
Welker, T.L., Overturf, K.E., Abernathy, J.W. 2019. Effect of aeration and oxygenation on growth and survival of rainbow trout in a commercial serial-pass, flow-through raceway system. Aquaculture Report. 14:1-9. https://doi.org/10.1016/j.aqrep.2019.100194.
Zhao, H., Fuller, S.A., Thongda, W., Abernathy, J.W., Beck, B.H., Peatman, E. 2018. SNP panel development for genetic management of wild and domesticated white bass (Morone chrysops). Animal Genetics. 50(1):92-96. https://doi.org/10.1111/age.12747.
Beck, B.H., Aksoy, M., Shoemaker, C.A., Fuller, S.A., Peatman, E. 2019. Antimicrobial activity of the biopolymer chitosan against Streptococcus iniae. Journal of Fish Diseases. 42:371-377.
Green, B.W., Rawles, S.D., Schrader, K.K., Gaylord, T.G., McEntire, M.E. 2019. Effects of dietary protein content on hybrid tilapia (Oreochromis aureus×O. niloticus) performance, common microbial off-flavor compounds, and water quality dynamics in an outdoor biofloc technology production system. Aquaculture. 503:571-582. https://doi.org/10.1016/j.aquaculture.2019.01.034.
Green, B.W., Schrader, K.K., McEntire, M.E. 2018. Effects of solids removal on water quality and channel catfish production in a biofloc technology production system. Journal of Applied Aquaculture. 31(1):1-16. https://doi.org/10.1080/10454438.2018.1536010.
Roy, L., Rawles, S.D., Kelly, A., Stone, N., Park, J., Webster, C.D. 2019. The effects of different winter feeding regimens on growth, survival, and fatty acid composition of fathead minnow and golden shiners. North American Journal of Aquaculture. 81:189-200. https://doi.org/10.1002/naaq.10086.
Hagar, J., Rawles, S.D., Xiong, Y., Newman, M., Thompson, K., Webster, C.D. 2019. Listeria monocytogenes is inhibited on fillets of cold-smoked sunshine bass, Morone chrysops x M. saxatilis, with an edible corn zein-based coating incorporated with lemongrass essential oil or nisin. Journal of the World Aquaculture Society. 50(3):575-592. https://doi.org/10.1111/jwas.12573.
Roy, L., Rawles, S.D., Kelly, A., Stone, N., Haukenes, A., Webster, C.D. 2019. Feeding regime has little impact on fatty acid composition and weight loss of hybrid crappie, Pomoxis nigromaculatus x Pomoxis annularis, and redear sunfish, Lepomis microlophus, at low (winter) water temperatures. North American Journal of Aquaculture. 81(2):140-152. https://doi.org/10.1002/naaq.10081.