Non Technical Summary
As the aquaculture industry expands in the United States, investigation of the commercial aquaculture potential of new species is important as this may allow producers to operate in new locations and would provide new products for market. Burbot is a fish that demonstrates potential for commercial production in northwestern states. All life stages of this cool to cold-water species can be grown in captivity and production conditions are shared with rainbow trout. Burbot offers a thick flake white fillet, delicacies such as roe and liver, and leather. Finally, burbot is resistant to many salmonid pathogens, offering an option to add the species to existing trout production operations. This proposed independent research project will use recently published methods developed for burbot and other farmed fish species to describe egg incubation and larval culture in recirculating aquaculture systems and investigate development of the immune system. The specific objectives for this "Advancing Science" EWD Fellowship project: 1) characterize egg survival and incubator microbiomes within recirculating and flow through systems, 2) characterize larval survival, development, and culture tank microbiomes within recirculating and flow through systems, and 3) investigate the development of immune organs and proteins associated with adaptive immunity in larval diploid and triploid burbot. The results from this project will be of interest to commercial cold-water aquaculture producers and agencies using burbot for management applications.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
307	3719	1060	75%
305	3719	1090	25%

Knowledge Area
305 - Animal Physiological Processes; 307 - Animal Management Systems;

Subject Of Investigation
3719 - Other cultured finfish;

Field Of Science
1090 - Immunology; 1060 - Biology (whole systems);

Goals / Objectives
Major goals:This project will improve the understanding of methods forearly life cultureof burbot (Lota lota), potentially reducing total waterconsumption withoutcompromising production. Additonally, this project will yeild a developnmetal timefline for immunological development in diploid and triploid burbot, potentially improving vaccination effcicay. Finaly, the overarching goal is to improve the understanding of burbot aquaculture to increasepotential for commercial development of burbot as a food fish.Objectives:1. Characterize survival of burbot eggs and incubator microbiomes within flow-through and recirculating aquaculture incubation systems.2. Characterize larval development and survival, and tank microbiome, within flow-through and recirculating aquaculture systems.3. Characterize development of immunological organs in diploid and triploid burbot.

Project Methods
Objective 1. Survival of burbot eggs (from fertilization to hatch), and incubator microbiomes will be characterized for groups of eggs incubated in recirculating and flow-through aquaculture systems. Utilizing the captive burbot broodstock housed at the UI Aquaculture Research Institute (UI-ARI), three distinct family groups will be created, each family group generated from eggs of a single female fertilized by milt of two males; parental fish will be spawned once and not incorporated into multiple family groups. After egg hardening, 50 minutes post fertilization, three replicates of ~5ml of fertilized eggs will be collected from each family group and placed in separate incubator tubes within each of the treatments, for the duration of incubation (~6 weeks). Six treatments will be utilized: 1) flow-through incubation, 2) flow-through incubation with hydrogen peroxide treatment (500ppm for 15 minutes, once daily) 3) flow-through incubation with UV treatment 4) recirculating incubation 5) recirculating incubation with hydrogen peroxide treatment (500ppm for 15 minutes, once daily), and 6) recirculating incubation with UV treatment. The recirculating aquaculture systems will be seeded approximately two months prior to initiation of the experiment by addition of 18g formulated fish feed and 12mg NH4-NL-1d-1. All eggs will be incubated at ~2.0°C and incubator flows will facilitate water turnover every 10 minutes. To determine survival, initial egg volumes will be collected at stocking and again one day prior to hatch. Sampling to investigate microbiome will be conducted weekly, beginning one week prior to stocking and ending a week after hatch. Each microbiome sample will consist of 100ml of water collected from each incubator and inflowing water. Microbiome samples will be used to determine colony forming units (25ml) and total number of bacteria (25ml) as described in Attramadal et a. (2012). Additionally, one hour after collection, 50ml of microbiome samples will be centrifuged at 20,000g for 10 minutes, decanted, and the pellet resuspended in 40ml of 95% molecular grade ethanol and stored until use. At the completion of the trial, DNA will be extracted from the microbiome samples using a commercially available kit (Qiagen Inc.) and stored at -80°C until shipped to a commercial laboratory for genomic analysis (Research and Testing Laboratories, Lubbock, TX). The genomic analysis will be performed using a targeted 16s approach (V3, V4) and subsequent pipeline to identify operational taxonomic units (OTUs). The contracted laboratory will identify these species using Next-generation sequencing (NGS), outputs will be analyzed by PD Oliver at the Hagerman Fish Culture Experiment Station (Hagerman, ID). The microbiota profiles will be compared and interpreted to determine the compositional changes related to the incubation method. Objective 2. Survival of burbot larvae, from 5 days post hatch (dph) until complete metamorphosis to the juvenile stage (~ 77 days), and culture tank microbiomes will be characterized for groups of larvae cultured in recirculating and flow-through aquaculture systems. Larvae will be obtained from the UI-ARI and stocked, at 400 individuals L-1, into identical 4L culture tanks (8-15°C; >6.0mg L-1 dissolved oxygen, 1 hour water turnover). Six treatments will be utilized: 1) flow-through incubation, 2) flow-through incubation with hydrogen peroxide treatment (500ppm for 15 minutes, once daily) 3) flow-through incubation with UV treatment 4) recirculating incubation 5) recirculating incubation with hydrogen peroxide treatment (500ppm for 15 minutes, once daily), and 6) recirculating incubation with UV treatment. Mature recirculating aquaculture systems will be generated as described in Objective 1. Culture tanks will be organized into a randomized incomplete block design, each of the three blocks will contain six culture tanks representing one replicate of each of the treatments. Larvae will be fed according to established burbot larval culture methods.