Recipient Organization
EVERGREEN AQUATICS
12619 NE 359TH ST
LA CENTER,WA 986293635
Performing Department
(N/A)
Non Technical Summary
Burbot is a novel candidate for commercial aquaculture production, it grows well in captivity, its fillets,liver, and roe are highly regarded for human consumption, and its basic husbandry requirements areunderstood. New aquaculture species are necessary to promote aquaculture expansion that willcontribute to increased food security and economic growth in the US. The early life stages of this speciesare associated with relatively high mortality, the research proposed here will improve survival andviability of this species through these stages and allow for consistent, reliable production of young fish ina hatchery setting. If this research is a success, hatcheries producing young burbot may have anenhanced ability to supply farms with fish at a stage where survival is generally high, and this interactionmay benefit many aquaculture farmers and the communities they share.Burbot research conducted at the University of Idaho and Evergreen Aquatics will use larval and juvenileburbot to determine if survival and production can be enhanced through modification of egg fertilizationmethods, larval water quality conditions, and novel vaccine development. The information that islearned from this project will be made available to the public, specifically the industrial and scientificcommunities, interested in producing this species. This project is intended to promote the commercialproduction of burbot, to provide farmers and consumers with an additional agricultural species forproduction and human consumption.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Goals / Objectives
This project aims to increase the efficacy of burbot (Lota lota) commercial aquaculture, with theultimate goal of establishing a commercial burbot production hatchery to supply burbot to the USmarket. To increase efficacy of commercial burbot aquaculture, three objectives are proposed:1. Development and evaluation of a vaccine to reduce mortality from bacterial pathogens infingerling and juvenile burbot.2. Investigation of parameters that may cause swim bladder overinflation related mortality inlarval burbot.3. Determination of conditions that result in reliable and consistent tetraploid burbot production.
Project Methods
Vaccine development:To produce an immersion vaccine/bacterin, Aeromonas sp. A141 will be grown in culture media (trypticsoy broth; TSB) for 48-72h and killed with formalin (0.5% v/v), prior to sterility assessment via plateculture. Since a challenge model was developed in larger fish (~40 g), it will be important to determinean LD50 for burbot in the 4-5 g size range. This will be accomplished by both immersion in a solution oflive bacteria (dose range from 1x104 CFU/ml to 1x108 CFU/ml) and by intraperitoneal (IP) injection of100µl/fish of cultures ranging from 1x104 CFU/ml to 1x108 CFU/ml. All challenges will be conducted inthe College of Natural Resources Aquatic Animal Laboratory (AAL), a biosafety level II facility. Once anappropriate challenge model and the dose are developed, burbot will be vaccinated by immersion withthis killed bacterin containing approximately 1x108 killed bacterial cells/ml. Fish (between 1 and 2 g)will be given a primary vaccination and a booster vaccination 2 weeks later. At 8 weeks post primaryvaccination, the burbot will be challenged with the virulent Aeromonas sp. isolate (A141) and placed intriplicate tanks of 25 fish per tank. Vaccinated control fish will receive an IP injection of the bacterin, andmock controls will also be challenged using culture media (tryptic soy broth).The analysis will include necropsy and re-isolation of Aeromonas sp. on 10% of mortalities in each tankeach day; polymerase chain reaction (PCR) will be used to confirm etiological agent from all re-isolatedsamples. Serum samples will be collected one day before vaccination, booster, challenge, and at theendpoint of the challenge; all serum samples will be analyzed via enzyme-linked immunosorbent assay(ELISA) to determine anti-Aeromonas sp. specific antibodies, see description below in "Related Researchor Research and Development" section. Endpoint cumulative percent mortality (CPM) at 21 days post-challenge (dpc) will be determined, and mortality of vaccinated fish will be compared to unvaccinatedcontrols (immersed in TSB alone) to determine Relative Percent Survival (RPS). Statistical significancebetween treatments will be determined by a one-way analysis of variance (ANOVA) followed by aTukey's posthoc test.Should this immersion vaccine elicit a protective immune response following laboratory trials, then fieldtrials at Evergreen Aquatics will be initiated (likely as part of a Phase II project) to determine benefitsunder production conditions. This portion of the project was included as part of this Phase I applicationbecause, if successful, it could represent a primary product that would contribute to commercial-scaleproduction of this species. With the initial work showing proof of concept for vaccination in this fishspecies via injection, an immersion-based vaccine would represent a further tool to improve aquaticanimal health management for aquaculture.Swimbladder overinflation:To address the issue of swim bladder over-inflation in larval burbot a single experiment will beconducted during phase I of this project. Larvae burbot, approximately 0dph, will be stocked into ninereplicate 4L tanks at 400 larvae/L. Larvae will be cultured according to standard procedures developedat UI, outlined in the "Related Research or Research and Development" section of this document. ThreeC02 concentrations will be maintained 10, 27.5, and 45mg/L, these concentrations are based onsalmonid aquaculture and previous observations of burbot aquaculture (Rekecki et al. 2016; Good et al.2010). The experiment will be terminated when larvae in all replicates are weened onto a commercialmicroparticulate diet. Photographs will be taken every three days through the experiment, for each tankthree groups of approximately 10 larvae will be randomly collected and photographed. Each group of 10larvae will be placed in a single well of a 12-well plate, dewatered, photographed, and returned to therespective tank. Furthermore, the surface of every tank will be photographed every day and analyzed, ata later date, to determine number of over-inflated individuals. At the day of experiment termination,larvae will be counted in each tank to determine survival, 10% of surviving larvae will be humanelysacrificed, photographed and then preserved for dry weight analysis.Phase I experimental data will be analyzed via one-way analysis of variance followed by a Tukey's test todetermine significant treatments, after determination of data normality and homogeneity of variance.Non-normal data will be analyzed using a Kruskal-Wallis test followed by a Dunn's test to determinesignificant treatments. For either normal or nonnormal data, significance will be confirmed when p-value ≤ 0.05.Tetraploid Induction:To induce tetraploidy in recently fertilized eggs a shock is applied to prevent the first cellular division, tostandardize shock timing the percent of time leading to first division is used. There will be ninetreatments to attempt to identify the ideal timing for application of shock to recently fertilized eggs inorder to induce cellular chromosome duplication, control (no shock), 60, 65, 70, 75, 80, 85, 90, and 95%of first cellular division. Batches of eggs from three females will be used to replicate the treatments,approximately 2.5mL of eggs will be used for each replicate; 1mL of burbot eggs is equal toapproximately 1000 eggs. Larvae from each replicate, 10 larvae, will be humanely sacrificed and used forploidy analysis.Ploidy data will be analyzed via one-way analysis of variance followed by a Tukey's test to determinesignificant treatments, after determination of data normality and homogeneity of variance. Non-normaldata will be analyzed using a Kruskal-Wallis test followed by a Dunn's test to determine significanttreatments. For either normal or nonnormal data, significance will be confirmed when p-value ≤ 0.05.