DISEASE RESISTANCE TRADE-OFFS AND THE PERFORMANCE OF SELECTIVELY-BRED OYSTERS FACING ENDEMIC DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1011778
• Grant No.: 2017-67012-26127
• Cumulative Award Amt.: $152,000.00
• Proposal No.: 2016-04884
• Project Start Date: Feb 15, 2017
• Project End Date: Feb 13, 2020
• Grant Year: 2017
• Program Code: [A7201]- AFRI Post Doctoral Fellowships

Recipient Organization
UNIVERSITY OF RHODE ISLAND
19 WOODWARD HALL 9 EAST ALUMNI AVENUE
KINGSTON,RI 02881

Performing Department
Fish-Animal-Vet-Science

Non Technical Summary
Oyster farming is a $300 million industry in the United States, supporting thousands of small farms and sustainable jobs in coastal regions. While growing, industry yields are severely constrained by a number of naturally-occurring infectious diseases that impact the growth and survival of farm-raised oysters. Dermo disease, a fatal condition caused by the naturally-occurring pathogen Perkinsus marinus, is among the most impactful along the Atlantic and Gulf coasts, and developing selectively-bred oyster lines that perform well in the face of Dermo disease is a high priority for hatcheries and breeding programs. The goal of this project is to identify disease defense traits to target for selective breeding. This research project combines controlled laboratory experiments testing how selectively-bred oysters resist and tolerate exposure to Perkinsus marinus with a field study testing how pathogen resistance and pathogen tolerance benefit the yield of farm-raised oysters. The ultimate goal of this work is to develop simple tests of optimal disease defense traits in selectively-bred oyster lines that can be readily integrated into oyster breeding programs.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	0811	1170	25%
311	0811	1070	25%
311	0811	1081	25%
311	0811	1110	25%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
0811 - Shellfish;

Field Of Science
1070 - Ecology; 1081 - Breeding; 1110 - Parasitology; 1170 - Epidemiology;

Keywords

aquaculture

oyster

disease

resistance

Goals / Objectives
The growth and sustainability of the oyster aquaculture industry is constrained by a number of naturally-occurring infectious diseases that impact the growth and survival of farm-raised oysters. Dermo disease, a fatal condition caused by the naturally-occurring pathogen Perkinsus marinus, is among the most impactful oyster diseases along the Atlantic and Gulf coasts, and developing selectively-bred oyster lines that perform well in the face of Dermo disease is a high priority for hatcheries and breeding programs. The goal of this project is to identify disease defense traits to target for selective breeding. Traditionally, the ability for hosts to defend against disease has been thought to be a result of immune system functions that detect and eliminate invading pathogens. However, immunity has often shown to be unassociated from the ability for hosts to control and eliminate pathogens, suggesting that immunity can also be a function of hosts' ability to tolerate the presence of pathogens. In principle, defense against infectious diseases can be divided into three conceptually different strategies: avoidance, or behaviors that reduce the risk of pathogen exposure, resistance, the immune functions and physiological processes that reduce pathogen burden once infections are established, and tolerance, or the ability for hosts to limit disease severity induced by established infection. In the case of oyster aquaculture faced with Dermo disease, should any of these defense strategies be targeted over others? Oysters rely on a repertoire of avoidance, resistance, and tolerance strategies to protect against the impact of Dermo disease, and the objectives of this project are to move toward the development of oyster lines capable of producing high yields in the face of Dermo disease by testing:How the relative contribution of avoidance, resistance, and tolerance strategies covary among selectively-bred oyster families exposed to the agent of Dermo disease, andThe relative contribution of these co-varying strategies to the performance and yield of farm-raised oysters.

Project Methods
Selectively-bred oyster families I will obtain 50 selectively-bred oyster families (N = 1000 individuals from each family) from the Aquaculture Genetics and Breeding Technology Center at the Virginia Institute of Marine Science under an ongoing material transfer agreement between ABC and my primary mentor, Dr. Dina Proestou. The oyster families will be spawned from brood stock that have demonstrated to vary in their response to Dermo disease. All oyster families will be transported to the Blount Aquaculture Research Laboratory at the University of Rhode Island and placed in individual silos housed within a closed, recirculating upweller system. The upweller system will receive 1μm filtered UV-sterilized seawater and the oysters will be fed the cultured algae Isochrysis galbana, Pavlova lutheri, and Chaetoceros calcitrans. This system is designed to grow oysters in quarantine, eliminating exposure to Perkinsus marinus, the agent of Dermo disease, until challenge experiments. When oysters reach a mean shell length of approximately 25mm, I will censor ten oysters from each family (N = 500 oysters), and the censored oysters will be shucked and their tissues placed in 70% ethanol. I will quantify tissue concentrations of P. marinus by quantitative PCR of extracted DNAs from the censored tissue samples to validate that oysters are free of the agent of Dermo disease prior to laboratory experiments.Objective 1: quantifying Dermo defense strategiesIndividual oysters (N = 200 from each family) will be placed in individual cups containing 500 ml sterile filtered seawater containing cultured I. galbana, P. lutheri, and C. calcitrans at a concentration of 105 algal cells ml-1. Each cup will receive an environmental P. marinus isolate (ATCC® strain 50509, "DBNJ"; American Type Culture Collection, Manassas, VA) at one of four doses: a no dose control, 105 cells cup-1, 106 cells cup-1, and 107 cells cup-1. I will allow the oysters to feed on the algae and P. marinus cocktail for six hours, when at this time, ten oysters from each family at each P. marinus dose will be censored and their tissues shucked and placed in 70% ethanol. The remaining oysters will be rinsed in sterile seawater and moved to replicate flow-through seawater systems receiving 1μm filtered, UV-sterilized seawater. All systems will be fed cultured I. galbana, P. lutheri, and C. calcitrans daily, and the tanks will be rinsed daily to remove accumulated biodeposits. I will check for oyster mortality daily and all observed mortalities will be removed from the system and their tissues shucked and placed in 70% ethanol. An additional ten oysters from each family at each dose will be censored at one, two, three, seven, fifteen, 30, and 60 days after the initial P. marinus exposure, and all the remaining oysters will be censored 90 days after P. marinus exposure. I will quantify tissue concentrations of P. marinus from DNA extracted from the censored tissue samples, as well as from DNA extracted from the tissue samples taken from the observed mortalities, by quantitative PCR. I will use the tissue concentrations of P. marinus sampled six hours after initial exposure as a measure of pathogen avoidance, testing for differences among the oyster families and by dose using analysis of covariance. I will test for differences in tissue concentrations of P. marinus among the oyster families and across all P. marinus doses at all sampling times using hierarchical linear model. The fitted coefficients quantifying the effect of oyster family in the hierarchical model will be used as a relative measure of pathogen resistance. I will quantify pathogen tolerance for each oyster family as the slope of the fitted linear relationship between a) the six-hour tissue concentrations of P. marinus across all four P. marinus doses for each family and b) the estimated 90-day Kaplan Meier survival probability for each family at each dose. Objective 2: field evaluation of oyster performanceThe remaining oysters in the recirculating upweller system (N ≈ 800 from each family) will be measured and weighed and then deployed by family in individual mesh bags to one of three sites in Rhode Island where Dermo disease is present (N = 200 from each family at each site): Ninigret Pond, Winnapaug Pond, or Quonochontaug Pond. The sites will be visited bi-monthly for eighteen months. For each sampling time, all mortalities will be removed and the remaining oysters will be measured, weighed and sorted into new mesh bags. All oysters will be removed following the eighteen-month sampling event. I will use total yield, the product of survival and growth, to quantify the performance of the oyster families at each site. I will test for differences in yield among the families, given each family's measured P. marinus avoidance, resistance, and tolerance from the laboratory experiments, using a mixed effects regression correcting for differences among the sampling times and sites by treating these independent variables as random effects.Efforts to cause change in knowledgeI will work closely with the Aquaculture Genetics and Breeding Technology Center at the Virginia Institute of Marine Science throughout all aspects of the project and beyond to develop tools to incorporate measures of disease defense into a multi-trait breeding program for oysters. The results from this project will be used directly to identify key disease defense traits for selective breeding programs to target.How the outputs will be evaluatedI will evaluate progress toward the project's core career development and mentoring objectives and project milestones through weekly informal meetings with my primary mentor and all involved undergraduate and graduate students, and monthly targeted project meetings with my primary and collaborating mentors. The career development and mentoring objectives are best met through the effective execution of the research plan. With all research, pitfalls arise and problems are encountered that can delay results or require an alternative experimental approach. I expect this to be the case and have planned and budgeted flexible time to repeat or modify my experimental design.

Progress 02/15/17 to 02/14/20

Outputs
Target Audience:This project involved and interacted with oyster aquaculture industry groups, including nursery and hatchery operators, state and federal aquaculture regulators, including the Rhode Island Department of Environmental Management, North Carolina Division of Marine Fisheries, and USDA Animal and Plant Health Inspection Services, and the scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has allowed me to independently establish myself as a leading shellfish pathologist and disease ecologist. I have led workshops, interacted with state and federal aquaculture regulators, and collaborated with industry. My training and development through the NIFA postdoctoral fellowship program prepared me for my current position as an assistant professor of shellfish pathology at North Carolina State University and director of the shellfish pathology program for the state of North Carolina. How have the results been disseminated to communities of interest?This project has given me the opportunity to open discussions with the oyster aquaculture industry in Rhode Island and throughout the Atlantic coast. I have met with oyster hatchery managers in Virginia, New Jersey, North Carolina, and Rhode Island, and visitied oyster farm sites throughout New England and the Atlantic and Gulf coasts. I have published peer reviewed articles in international journals and presented project updates and results from the laboratory and modeling work at invited seminars at the University of Southern Mississippi Gulf Coast Research Laboratory, Northeastern University's Marine Science Center, the Virginia Institute of Marine Sciences, the University of North Carolina Institute of Marine Science, the North Carolina State, University Center for Marine Science and Technology. In addition, I have expanded my service and outreach responsibilities to my field by taking on the role of board president of the East Coast Shellfish Research Institute, and organization formed to support shellfish aquaculture production and sustainabilty through research, education, and outreach activities. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? I conducted all laboratory experiments addressing these project goals. There are numerous examples, including some published through the course of this fellowship, of selectively-bred oysters resisting infection with disease-causing parasites and tolerating disease impacts by continuing to survive even when infected. Results from the laboratory experiments conducted as part of this NIFA postdoctoral fellowship suggest that resistance to the oyster parasite Perkinsus marinus covaries with tolerance to impacts from infection, a condition known as dermo disease. That is, selectively bred oysters that survive and perform well when faced with dermo disease do little to resist infection. Follow-up work integrating these covarying traits into epidemiological models that extend individual impacts to entire oyster populations has demonstrated a counterintuitive outcome: when aquaculture production relies on oysters bred to tolerate disease but not resist infection, disease prevalence and its impact increase in cultured oyster populations and to any wild oyster populations nearby. With my primary and collaborating mentors I have secured follow on funding to address this new hypothesis of disease risks facing the oyster aquaculture industry.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Cantrell, D.L., M.L. Groner, T. Ben-Horin, J. Grant, C. Revie. 2020. Modeling pathogen dispersal in marine fish and shellfish. Trends in Parasitology. In Press.
• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Aalto, E., K. Lafferty, S. Sokolow, R. Grewelle, T. Ben-Horin, C. Boch, P. Raimondi, S. Bograd, E. Hazen, M. Jacox, F. Micheli, G.A. De Leo. 2020. Models with environmental drivers offer a plausible mechanism for the rapid spread of infectious disease outbreaks in marine organisms. Scientific Reports.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2020 Citation: Levine, J., J.M. Law, and T. Ben-Horin. 2020. Bivalve Medicine. In: G.A. Lewbart (Ed.) Invertebrate Medicine. John Wiley & Sons, Inc.

Progress 02/15/17 to 02/13/20

Outputs
Target Audience:This project involved and interacted with oyster aquaculture industry groups, including nursery and hatchery operators, state and federal aquaculture regulators, including the Rhode Island Department of Environmental Management, North Carolina Division of Marine Fisheries, and USDA Animal and Plant Health Inspection Services, and the scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has allowed me to independently establish myself as a leading shellfish pathologist and disease ecologist. I have led workshops, interacted with state and federal aquaculture regulators, and collaborated with industry. My training and development through the NIFA postdoctoral fellowship program prepared me for my current position as an assistant professor of shellfish pathology at North Carolina State University and director of the shellfish pathology program for the state of North Carolina. How have the results been disseminated to communities of interest?This project has given me the opportunity to open discussions with the oyster aquaculture industry in Rhode Island and throughout the Atlantic coast. I have met with oyster hatchery managers in Virginia, New Jersey, North Carolina, and Rhode Island, and visitied oyster farm sites throughout New England and the Atlantic and Gulf coasts. I have published peer reviewed articles in international journals and presented project updates and results from the laboratory and modeling work at invited seminars at the University of Southern Mississippi Gulf Coast Research Laboratory, Northeastern University's Marine Science Center, the Virginia Institute of Marine Sciences, the University of North Carolina Institute of Marine Science, the North Carolina State, University Center for Marine Science and Technology. In addition, I have expanded my service and outreach responsibilities to my field by taking on the role of board president of the East Coast Shellfish Research Institute, and organization formed to support shellfish aquaculture production and sustainabilty through research, education, and outreach activities. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? I conducted all laboratory experiments addressing these project goals. There are numerous examples, including some published through the course of this fellowship, of selectively-bred oysters resisting infection with disease-causing parasites and tolerating disease impacts by continuing to survive even when infected. Results from the laboratory experiments conducted as part of this NIFA postdoctoral fellowship suggest that resistance to the oyster parasite Perkinsus marinus covaries with tolerance to impacts from infection, a condition known as dermo disease. That is, selectively bred oysters that survive and perform well when faced with dermo disease do little to resist infection. Follow-up work integrating these covarying traits into epidemiological models that extend individual impacts to entire oyster populations has demonstrated a counterintuitive outcome: when aquaculture production relies on oysters bred to tolerate disease but not resist infection, disease prevalence and its impact increase in cultured oyster populations and to any wild oyster populations nearby. With my primary and collaborating mentors I have secured follow on funding to address this new hypothesis of disease risks facing the oyster aquaculture industry.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Aalto, E., K. Lafferty, S. Sokolow, R. Grewelle, T. Ben-Horin, C. Boch, P. Raimondi, S. Bograd, E. Hazen, M. Jacox, F. Micheli, G.A. De Leo. 2020. Models with environmental drivers offer a plausible mechanism for the rapid spread of infectious disease outbreaks in marine organisms. Scientific Reports.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2020 Citation: Levine, J., J.M. Law, and T. Ben-Horin. 2020. Bivalve Medicine. In: G.A. Lewbart (Ed.) Invertebrate Medicine. John Wiley & Sons, Inc.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Cantrell, D.L., M.L. Groner, T. Ben-Horin, J. Grant, C. Revie. 2020. Modeling pathogen dispersal in marine fish and shellfish. Trends in Parasitology. In Press.

Progress 02/15/18 to 02/14/19

Outputs
Target Audience:The project goals and outcomes target the oyster aquaculture industry including hatcheries and growers, and the scientific community supporting this industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has allowed me to independently establish myself as a leading shellfish pathologist and disease ecologist. I led a workshop at the Virginia Institute of Marine Sciences to further develop mathematical models evaluating interactions between open-water oyster aquaculture and wild oyster populations through shared parasites. This research has led to four competitive grant proposals submitted over the reporting period, one of which was funded. In addition to grant writing and my development as an independent scholar, I have expanded my service responsibilities to my field. I have taken on the role of board president of the East Coast Shellfish Research Institute, and organization formed to increase public awareness of shellfish aquaculture activities through research, educational and informational activities. How have the results been disseminated to communities of interest?This project has given me the opportunity to open discussions with the oyster aquaculture industry in Rhode Island and throughout the Atlantic coast. I have met with oyster hatchery managers in Virginia, New Jersey, and Rhode Island, and visitied oyster farm sites throughout New England and the mid-Atlantic and Gulf coasts. I have presented project updates and results from the modeling work at invited seminars at the University of Southern Mississippi Gulf Coast Research Laboratory, Northeastern University's Marine Science Center, the Virginia Institute of Marine Sciences, and the North Carolina State University Center for Marine Science and Technology. What do you plan to do during the next reporting period to accomplish the goals?I completed all proposed experiments over this reporting period. Over the next reporting period I will complete all data analysis and prepare manuscripts for publication in peer-reviewed journals. I plan to give a final project update and the meeting of the Coastal and Estuary Research Foundation in Mobile, AL in the fall of 2019.

Impacts
What was accomplished under these goals? I conducted all laboratory experimentes quantifying how traits covary in oyster families and which traits or combination of traits contribute most to the performance and yield of farm-raised oysters.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Ben-Horin, T., S.K. Allen Jr., J.M. Small and D.A. Proestou. 2018. Genetic variation in anti-parasite behavior in oysters. Marine Ecology Progress Series. 594: 107-117.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Ben-Horin, T., C.A. Burge, D. Bushek, M.L. Groner, L. Huey, D.A. Proestou, G. Bidegain, and R.B. Carnegie. 2018. Intensive oyster aquaculture can reduce disease impacts on sympatric wild oysters. Aquaculture Environment Interactions. 10: 557-567.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2019 Citation: Ben-Horin, T., G. Bidegain, G.A. de Leo, E.E. Hofmann, M.L. Groner, H. McCallum and E.N. Powell. Modeling disease in the sea. In: D.C. Behringer, K.D. Lafferty and B.R. Silliman (Eds.) Marine Disease Ecology. Oxford University Press.
• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Proestou, D.A., R.J. Corbett, T. Ben-Horin, J.M. Small and S.K. Allen Jr. Defining Dermo resistance phenotypes in an eastern oyster breeding population.

Progress 02/15/17 to 02/14/18

Outputs
Target Audience:The project goals and outcomes target the oyster aquaculture industry including hatcheries and growers, and the scientific community supporting this industry. Changes/Problems:No major changes, but our first oyster families failed to set in the spring of 2017. Instead, a second round of oyster families was set in the summer of 2018. This gave a short delay to the proposed experiments, but this allowed me to refine the experimental design with a pilot experiment and fine tune the methods to quantify disease defense traits. All ongoing experiments are on track for successful completion. What opportunities for training and professional development has the project provided?This project has given me the opportunity to establish myself as an authority in the field of marine disease ecology, with respect in particular to how wild and open-water-cultured populations interact through shared water-borne parasites. As part of this research thrust, I took part in a NSF-funded workshop aiming to understand and improve the management of diseases impacting marine ecosystems, sponsored by the NSF Research Coordination Network "Evaluating the Impacts of a Changing Ocean on Management and Ecology of Infectious Marine Diseases" (NSF OCE Award # 1215977). I took on a leadership role in this project where I developed novel mathematical models to evaluate interactions between open-water aquaculture and wild populations through shared parasites. This research has led to a peer-reviewed publication, and new lens to view the epidemiological consequences of selective breeding for disease in aquaculture. This in turn has led to a new course of research, including a recently submitted research proposal to Rhode Island Experimental Program to Stimulate Competitve Reseach (EPSCoR) and an agenda to develop a broader course of research in a proposal to be submitted to the NSF-USDA Ecology of Infectious Diseases grant program. How have the results been disseminated to communities of interest?This project has given me the opportunity to open discussions with the oyster aquaculture industry in Rhode Island and throughout the Atlantic coast. I have met with oyster hatchery managers in Virginia, New Jersey, and Rhode Island, and visitied oyster farm sites throughout New England and the mid-Atlantic and Gulf coasts. I have presented project updates and results from the modeling work at the annual meeting of the National Shellfisheries Association in Knoxville, TN; the Ecology and Evolution of Infectious Diseases meeting in Santa Barbara, CA; and the annual meeting of the Coastal Foundation in Providence, RI. I was also invited to give the plenary symposium at the Southeast Society of Parasitologists in Charleston, SC, where I disseminated results from this work as part of a presentation on the ecology of infectious marine diseases. What do you plan to do during the next reporting period to accomplish the goals?All proposed experiments will be completed over the next reporting period and the results from these experiments will be synthesized and submitted to peer-reviewed journals. I plan to give a project update at theEcology and Evolution of Infectious Diseases meeting in Glasgow, Scotland in the summer of 2018 and a final project update at the annual meeting of the National Shellfisheries Association in the winter of 2019.

Impacts
What was accomplished under these goals? I conducted experiments to refine methods to quantify avoidance, resistance, and tolerance traits in selectively-bred oyster families. With these now demonstrated methods, the ongoing experiments are quantifying how these traits covary in oyster families and which traits or combination of traits contribute most to the performance and yield of farm-raised oysters.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Ben-Horin, T., S.K. Allen, J.M. Small and D.A. Proestou. In Press. Genetic variation in anti-parasite behavior in oysters. Marine Ecology Progress Series.
• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Ben-Horin, T. G. Bidegain, C.A. Burge, R.B. Carnegie, M.L. Groner, D.A. Proestou and D. Bushek. Interactions between wild and cultured populations sharing water-borne parasites.
• Type: Journal Articles Status: Submitted Year Published: 2018 Citation: Ben-Horin, T. and D. Bushek. Cooperative proliferation of the oyster parasite Perkinsus marinus.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2019 Citation: Ben-Horin, T., G. Bidegain, G.A. de Leo, E.E. Hofmann, H. McCallum and E.N. Powell. Modeling and forecasting disease dynamics in the sea. In: D.C. Behringer, K.D. Lafferty and B.R. Silliman (Eds.) Marine Disease Ecology. Oxford University Press.