Performing Department
(N/A)
Non Technical Summary
Background: Expansion of the US eastern oyster aquaculture industry depends on effective control of disease organisms encountered during oyster grow-out. Two pathogens, called dermo and MSX, are the cause of of significant mortality in wild and aquaculture oystersin the many states along the East coastand beyond, and as such, better disease-resistant stocks are required.Goals: The overall goal of this project is to develop a better understanding of therelationships between two significant oysterpathogens and performance of eastern oysters grown in the field destined for harvesting and sale.Specifically, an assessment of the heritability of resistance to each pathogen separately, or combined, will enable the development of improved disease-resistance breeding strategies.Objectives/Methods: The specific objectives of this study are tomeasure the variance insurvival of genetically-unique oyster groups infield trials at locations where the two diseases are naturally present in the environment. Unique genetic groups of oysters with a knownpedigree will be the foundational material for this work. The number of oystersthat get infected within each group as well as how severely theyare infected, including the severity of the hostresponse, will be studied using genetic techniques and techniques that allow microscopic examiniation of preserved oyster tissues. The relationship between the different genetic groups (pedigree information), field survival, growth and disease information will be combined using sophisticated mathematical modeling and predictive approaches.These results will clarify how the diseases effect oyster performance (correlation values) and how easily it is to breed for resistance to one or both pathogens (heritability values).Outcomes/Impacts: Upon completion of this research,the Aquaculture Genetics and Breeding Technology Center at VIMS willcreate new disease-resistant oyster stocks. These stocks will be distributed to to regional commercial hatcheries for propagation and sales of oyster larvae and seed with superior disease resistance. Improved oyster lines will increase the security and profitability of US oyster aquaculture production.
Animal Health Component
100%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
0%
Goals / Objectives
The over-arching goal of this project is to refine breeding techniques for eastern oysters for Perkinsus marinus (dermo) and Haplosporidium nelsoni (MSX) disease resistance and produce broodstock lines for the commercial oyster aquaculture industry that are highly resilient to both pathogens.The specific objectives of this study are the following:1) Assess survival of diploid eastern oyster families in field trials at locations where dermo and MSX are endemic2) Quantify prevalence and intensity of dermo and MSX disease within families using quantitative PCR and histology3) Assess genetic parameters, including heritability and correlation, of survival, disease and morphological traits using quantitative genetic models.
Project Methods
Methods/EffortsObjective 1 Spawning & Larval Husbandry - A subset of family material spawned in spring 2024 by ABC will be selected specifically for this project. Family crosses will be performed using a single female and single male only. Mantle tissue biopsies from all parents used will be preservedfor later genotyping and parentage confirmation. Standard ABC larval culture practices will be used to rear larvae through to setting.When oysters are large enough they will be transferred to the land-based upwelling system. Once the seed reaches 4-6 mm in shell length, seed from each family will bedeployed to the VIMS York River farm. Seed will be grown in mesh oyster bags in subtidal cages throughout the late summer and fall.Field Deployment - Beginning in March 2025, all families will be retrieved from the VIMS York River farm where a pre-sample of 30 individuals per family will be collected. Each oyster collected will be measured (length and whole weight taken) and shucked, with a piece of mantle tissue collected and preserved in 95% ethanol for downstream quantitative PCRanalysis. A histological cross section of each oyster will be taken, fixed and preserved in Davidson's solution for 48 hours followed by storage in 70% ethanol. Processing of histological samples will occur using methods below. After the samples have been taken, 300 oysters per family will be separated into three replicate adjustable long line baskets.These will be deployed on the ABC research farm in the York River,randomly distributing replicates across the adjustable long line farm. Six-hundred additional oysters per family, divided into three replicates, will be separated into three, 6mm mesh oyster bags and transferred to the farm owned by Cappahosic Oyster Company in the York River.Survival Assessment - In May, when H. nelsoni infection prevalence is typically the highest, at both field sites, the number of live oysters per replicate will be counted to assess survival. Ten oysters per replicatewill be removed at each sampling event. Length (greatest distance from hinge to bill) and whole weight will be recorded in for each oyster, then each oyster will be shucked, with a piece of mantle tissue and a histological section collected and preserved as above. In October, when P. marinus infection prevalence and intensity is typically the highest, at both field sites, the number of live oysters per replicate will be counted to assess survival. Ten oysters per replicatewill be removed at each sampling event. Samples will be cleaned as above and measured (length and whole weight taken) and shucked, with a piece of mantle tissue and a histological section collected and preserved as above. Environmental water quality data, including temperature, salinity, dissolved oxygen, and pH, will be collected at each site during each sampling event with a hand-held YSI.The field trial will be terminated in November at which time families at both field sites will be assessed for commercial trait metrics. Survival assessments will first be performed on all replicates, then 15 oysters will be randomly sampled per replicate for further measures.For all oyster samples, individual oyster total weight, length, width (greatest distance perpendicular to length) and height (depth) will be recorded. Whole weight will be recorded to the nearest tenth of a gram. Once external measurements are completed, oysters will be opened and all tissue removed. Individual meats will be allowed to drain of liquor briefly on a screen prior to recording meat weight. Mantle tissue from seven individuals per replicate will be excised and preserved in 95% ethanol for DNA preservation and down-stream genotyping.Objective 2Quantitative PCR - For each tissue sample preserved for qPCR, DNA will be extracted using Promega Wizard Genomic DNA Purification Kit. The extracted DNA will be assessed for assurance of quantity and quality and amounts standardized for qPCR analysis. Infection prevalence and intensity of P. marinus (dermo) and H. nelsoni (MSX) will be determined in each sample using a duplex qPCR assay on an ABI QuantStudio 3 Real-Time PCR System (DeFaveri et al. 2009, Wilbur et al. 2012).Histology - Histological samples will be processed through the VIMS Shellfish Pathology Lab. From each oyster to be assessed for P. marinus and H. nelsoni infection using histopathology, standard transverse sections including gills and mantle, gonad, stomach, intestine, digestive gland, and associated connective tissues will be taken and fixed in Davidson's solution (Shaw and Battle 1957), with subsequent processing for paraffin histology using standard methods. Six-micron, hematoxylin-and-eosin-stained slides will be evaluated on a compound microscope, with intensity of infection by either parasite, if detected, rated rare, light, moderate, or heavy using standard VIMS Shellfish Pathology Laboratory rubrics (Carnegie and Burreson 2011, Mann et al. 2014).Histological data will provide an additional understanding of host response relative to qPCR data collected for P. marinus and H. nelsoni. Cumulative survival per family and parasite load based on qPCR and histology results will be analyzed to assess the relationship between survival in the field and P. marinus and H. nelsoni infections. Linear mixed models will be used to model parasite prevalence with length, total weight, and meat yield to determine the impact of parasite infection on oyster growth.Objective 3Quantitative Genetic Analyses - Animal models will be used to partition the genetic variance from the total variance in disease acquisition among broodstock sources to estimate the heritability and correlation of traits associated with disease acquisition at each site. Trait data assessed will include survival, total weight, length, meat yield, and P. marinus and H. nelsoni prevalence and intensity (by qPCR) and by ordinal ranking of histopathological parasite observation.Data will be analyzed using ASReml to fit univariate or multivariate linear mixed models. Genetic parameters will be estimated for each trait at each site separately. Heritability will be calculated on both the observed and underlying scales to account for survival as a binary trait when evaluating selection across multiple traits (Dempster and Lerner 1950). Genetic correlations will be estimated from inter-trait variance and covariance structures.The various combinations of correlation analyses, along with the linear mixed modelling approaches described above, will serve to define the relationship between survival at these locations and the influence of P. marinus and H. nelsoni, by themselves or in combination. The correlation analyses will illustrate what type of parasite data is most informative and how best to incorporate it alongside commercial growth/shape traits so as to define a suitable weighted index to precisely breed for pathogen resistance while ensuring that commercially important traits like growth, shape or yield are not negatively impacted.Genotyping - Tissue samples collected from parents will be genotyped with a 66K high-density SNP genotyping array specifically designed for C. virginica to assess relatedness and genetic diversity. Tissue samples collected from oysters throughout the project will be selected from each site for genotyping with a low-density (3K) SNP array based on disease acquisition and morphometric traits. This genotype data will be used to confirm parentage, assess genomic associations with disease acquisition, perform a genome-wide-association study, and investigate pathogen detection by the SNP array.EvaluationThe impacts from this project will be evaluated directly by oyster growers seeking new disease-resistant lines. The quantity of new broodstock material distributed by ABC to commercial hatcheries and subsequent harvest and sales of this material will be a direct form of evaluation of the success and impacts of this research.