The aquatic biotechnology and genomics R&D strategy

Table of Contents

1. Introduction
2. Priority Research Themes
3. Vision for 2015
4. Issues, Trends, Drivers and Opportunities
   • 4.1 Canadian Trends and Initiatives
   • 4.2 The International Context
5. The Aquatic Biotechnology and Genomics R&D Strategy
   • Theme 1. Biotechnology and Aquatic Resource Management
   • Theme 2. Biotechnology and Aquatic Animal Health
   • Theme 3. Biotechnology and Aquatic Ecosystem Integrity
   • Theme 4. Novel Aquatic Animal Regulatory Science
6. Conclusion - Charting a Path Forward

The Aquatic Biotechnology and Genomics R&D Strategy

The following Strategy elaborates on the four themes and includes goals, objectives and actions designed to shape DFO's biotechnology and genomics. It is expected that the strategy will continuously evolve in response to departmental priorities, but that the broad themes and objectives capture key opportunities and activities that biotechnology and genomics applications and information can address, as it relates to DFO's mandate, strategic objectives and Science Sector's Renewal.

As biotechnology and genomics are enabling technologies and therefore multidisciplinary in nature, there are opportunities for the results from each of the research themes' activities and objectives to be incorporated and built on in other research themes. By mapping out the strategic direction and opportunities for biotechnology and genomics R&D, it is anticipated that new collaborations and partnerships can be identified, additional opportunities to transfer knowledge, expertise and applications will be realized, and efficiencies identified and implemented.

Theme #1: Biotechnology and Aquatic Resource Profiling

This research theme encompasses all activities related to understanding the genetic make-up of our aquatic resources. Biotechnology and genomics in this area include studying the genome of aquatic species, studying the population structure of these species and studying the genetics behind interactions between aquatic species and their environment (other species and environmental conditions).

Aquatic resource profiling directly supports sustainable fisheries, sustainable aquaculture, protection of biodiversity and recovery of species at risk. The goal is to optimize the productivity of the aquatic environment (from wild capture and aquaculture) while maintaining environmental health and biodiversity.

By charting each species, population by population, scientists can better assess which populations can support fisheries and how to prevent the loss of genetic diversity in designing breeding programs.

Endangered populations can also be identified and protected to ensure the genetic variability of each survives and thrives. Collated data on both endangered populations is housed in genomic libraries where the information is used to establish a clear understanding of population dynamics.

On the enforcement side, the development of forensic DNA capability in DFO has expanded the scope of enforcement actions while reducing expenditures associated with prosecutions for illegal harvest or sale of fish and shellfish.

Goal:

By 2015, to have developed biotechnology tools for genetic profiling of aquatic species and facilitated their widespread application in Canada and abroad, contributing to the sustainable use of aquatic resources.

Objectives:

1. Identify genetic markers to improve species, strain and stock identification for fisheries management and to allow for the protection and enhancement of biodiversity and aquatic fish habitat, including species at risk.
2. Improve biotechnology knowledge base for enhanced sustainability of aquaculture production: increase strain development and enhance biotechnology tools for identification and control of aquaculture species.
3. Enhance and apply research on population genetics and genomics to identify and monitor response of aquatic organisms due to environmental factors.

Objective #1

Identify genetic markers to improve species identification for fisheries management and to allow for the protection and enhancement of biodiversity and aquatic fish habitat.

Action #1:

Develop genetic markers for commercially important fisheries species to integrate into sustainable fisheries management practices.

• There is an increased demand for the "real-time" management of fisheries through stock identification techniques using genetic information derived from non-lethal sampling. This information and the speed at which it can be collected helps fishery managers decide if and when the fishery should open. This enables managers to avoid the harvest of populations of conservation concern, including endangered stocks.
• The US/Canada Coded Wire Tagging (CWT) program is expensive, slow and effectively tags only a fraction of the fish (hatchery only) caught in fisheries or recovered from the spawning grounds. Genetic markers, however, allow identification of different populations or groups whenever they become a conservation or management priority, replacing the need to tag the group years in advance of fishery analysis. This is of particular interest when the fisheries occur along migration routes used by mixed stock groupings.

Did You Know That…

DFO is using genomic tools such as mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) to identify stocks of beluga whales. The tools are used to estimate the proportion of beluga belonging to different stocks in an aboriginal mixed-stock fisheries and establish maximum harvest limits that reflect the sustainable harvest of each stock. Management actions are taken to enforce area closures when the DNA indicates that the area is frequented by a stock at a lower abundance level.

• For estimating population abundance, conventional mark and recapture techniques cannot be used for some species (e.g. rockfish die if brought to the surface for tagging). However, using genetic markers individuals can be genetically "marked" by creating a genetic profile from a non-destructive tissue sample (e.g., samples taken using a barbless hook) and then re-identified during the recapture process, either through resampling with barbless hooks or during a commercial or recreational fishery.
• Similarly, estimation of several cetacean population abundance is very difficult using conventional techniques as they occur over a wide geographical range, have clumped distribution and spend much time under water. Skin samples taken from various whale species including narwhal, beluga and bowhead are taken by aboriginal hunters via non-lethal sample harpoons. As well as being used for stock identification, these samples could be used to estimate the population number by mark and recapture estimation.

Did You Know That…

Due to its commercial viability and ease of tracking, salmon have become one of the most studied aquatic species in Canada and globally. Large, genetic baseline datasets for Pacific salmon, developed by DFO scientists, are used for the most intensive genetic management of fisheries on a real-time basis in the world. Over 10,000 chinook, sockeye and coho salmon samples are analyzed each year to manage fishery openings, enabling Canada to maximize catch under the US/Canada Pacific Salmon Treaty (PST) allocations while maintaining strict harvest limits on stocks of conservation concern. For example, real-time genetic management of the 2003 and 2004 north coast troll fishery on chinook salmon enabled the PST quota to be achieved for the first time since 1994 without overharvesting west coast Vancouver Island populations of conservation concern, resulting in increased annual revenue to the fisheries of over $3 million dollars.

Action #2:

Develop genetic markers for species of interest to Habitat Management including vulnerable species such as those listed by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC) for protection under the Species at Risk Act (SARA) or under the Convention on the International Trade of Endangered Species (CITES).

• DFO has developed tools that help the Department fulfill its responsibilities under the Species at Risk Act (SARA). Genetic methods are applied to identify population units deserving of designation (as vulnerable, threatened or endangered) according to COSEWIC.
• These methods are also used in developing a captive breeding program for Atlantic salmon; determining the establishment of Marine Protected Areas for marine organisms; and to investigate the effects of population bottlenecks and extensive transplantation on genetic diversity in salmon.

Did You Know That…

Using genetic profiling tools, DFO scientists have been able to distinguish the ‘inner' yelloweye rockfish population resident in the Strait of Georgia and Queen Charlotte Sound from an outer coastal population that extends from Oregon to Alaska. The genetic isolation and reduced abundance of the inner yelloweye rockfish population have been documented in a status report on the species for COSEWIC.

Action #3:

Enhance the use of forensic species identification for enforcement of fisheries and for the implementation of traceability requirements in other regulations.

• This action will build on the work done under Actions #1 and #2.
• The same genetic information that will support improved management decisions can also be used to increase the certainty and strength of the evidence base for prosecutions for the illegal harvest and sale of aquatic organisms.
• DFO also needs to enhance its ability to meet its CITES obligation to issue "non-detriment" permits for export of cultured product while ensuring that illegally harvested wild product is not laundered through the culture operations. This is required to ensure certainty for exporters of our products.

Action #4:

Expand the scope of genetics and genomics databases for species under DFO management and those aquatic species managed through international agreements.

• Information derived from DFO's genetics and genomics databases is used to manage domestic and international fisheries to enable harvest of abundant populations (species) while protecting "listed" populations and species of concern (e.g., species listed as endangered or threatened under the Species at Risk Act).

Did You Know That…

Molecular markers are being used to determine the population structure of redfish (Sebastes sp.) in the Northwest Atlantic. This information is particularly important given the transborder nature of these stocks. The use of microsatellite markers has highlighted the important role of hybridization that occurs between redfish species S. fasciatus, S. mentella, and S. marinus in the Gulf of St. Lawrence and Laurentian Channel. Population genetic structure and genetic diversity of these marine species are being determined, and the analysis of archived otoliths is providing key information on the distribution of these species as well as their recruitment history.

Objective #2:

Improve biotechnology knowledge base for enhanced sustainable aquaculture production: increase strain development and enhance biotechnology tools for identification and control of aquaculture fish.

Action #1:

Develop genetic markers to distinguish aquaculture strains from wild populations in order to assess their interactions.

• Genetic markers can be used for the accurate identification of escaped domesticated fish in the wild environment and to monitor their ecological and reproductive interactions with wild populations.
• Being able to identify aquaculture fish also enables the branding and tracing of aquaculture products, which is increasingly important as consumers demand more information and assurance on the source of seafood products.

Did You Know That...

Molecular markers are being used to study mollusc species such as giant scallop, soft shell clam, and blue mussels, in order to provide information to the aquaculture industry on the origin of collected spat, to help optimize production and to evaluate potential impacts of aquaculture practices on wild populations.

Action #2:

Incorporate genetic markers into pedigree identification of aquatic species and estimate the genetic merit in selective breeding programs.

• The identification of individuals to a family within a breeding program can be accomplished with genetic markers. This eliminates the need for rearing individual families in separate tanks and then applying tags for identification. The elimination of individual tanks saves hatchery costs and improves the estimation of "genetic merit" in breeding programs.
• Selective breeding may be revolutionized in near future by incorporating molecular markers into the identification of fish with superior performance traits, such as growth and survival, while they are still juveniles.
• Atlantic salmon aquaculture industries on both coasts rely on North American and European domesticated strains, respectively, with importation of new genetic material severely limited because of disease and ecological concerns. Productivity of both groups of fish will require constant improvement by selective breeding in order for the industries to remain competitive in the world market.

Did You Know That…

The development of Y-chromosome DNA markers associated with male sexual development have simplified the methods for production and maintenance of monosex salmon strains which have important benefits for production and conservation in aquaculture. For chinook salmon, monosex technology has been critical for the survival of the entire industry for more than 20 years; more recently, with Y-marker technology greatly simplifying the development of all-female monosex strains.

Action #3:

Develop methods of reproductive control.

• Various methods of reproductive control have and are continuing to be developed to avoid inter-breeding between aquaculture and wild fish populations that may have a negative impact on the wild fish. The effects of the reproductive control methods on the growth and survival of aquaculture strains will determine the feasibility of their implementation in aquaculture. Biotechnology and genomics tools can be used in the development and evaluation of reproductive control methods.
• Reproductive containment of cultured shellfish strains will become more important as more invertebrate species are cultured. Shellfish culture tends to be co-located with wild populations in the aquatic environment, increasing the opportunity and likelihood of reproductive interactions between cultured and wild populations.
• Research is also ongoing to evaluate the effectiveness of sterilization of male and female salmonids to prevent the breeding of escaped farmed salmon with wild salmon stocks.

Objective #3

Enhance and apply research on population genetics and genomics to identify and monitor responses of aquatic organisms to environmental factors.

Action #1:

Develop laboratory and bioinformatics capability for the application of cDNA microarrays and other genomics tools to detect physiological responses of aquatic organisms to environmental factors.

• Genomic and molecular biology technologies such as qPCR, Bacterial Artificial Chromosome (BAC) and Expressed Sequence Tag (EST) library screening and sequencing, and microarray analysis are being developed in-house and through partnership with large national and international collaborative efforts.
• DFO has begun to use these tools to determine the basis for altered behaviour and development (e.g., altered migration, reproduction and growth) and reduced survival due to environmental disruption. This information is becoming increasingly important for regulatory and management decisions in order to ensure sustainable use of aquatic resources. For example, applying DNA marker technology has allowed detection of endocrine disruption effects on salmon gonadal development caused by municipal and industrial effluents.
• Specialized genomics techniques include those that determine gene expression (i.e., when genes are ‘turned on'). Examples include quantitative PCR (qPCR), RNA isolation, microarray analysis, large-scale genome sequencing, genome-wide identification of important genes (QTLs), proteomics, metabolomics (the characterization of the metabolites in an organism), meta-genomics and analysis of genomes via bacterial artificial chromosome technology.

Theme #2: Biotechnology and Aquatic Animal Health

DFO contributes to the viability of our international seafood trade through the development and application of biotechnology tools to manage and protect aquatic animal health thereby enabling the Department to meet it's dual role in aquatic animal health: (1) to protect our aquatic ecosystems and (2) to meet the ever changing international standards, through the new National Aquatic Animal Health Program (NAAHP).

To control disease and its spread in aquatic animals, DFO scientists work with epidemiologists and veterinarians, deploying lab tests in commercial aquaculture settings and surveying wild stocks for diseases of concern. Quarantine and disease control measures are applied to aquaculture in order to preserve stocks and export trade. Diagnostic development, validation and application for reportable diseases is now administered through the new National Aquatic Animal Health Program (NAAHP), which involves the Canadian Food Inspection Agency (CFIA) and DFO.

These measures generate the knowledge to make recommendations in the management and control of significant aquatic animal diseases in Canada including economically important diseases like Infectious Salmon Anaemia (ISA) and Infectious Hematopoietic Necrosis (IHN) and the pathogen Haplosporidium nelsoni (MSX disease of oysters).

The health of aquatic animals is critical as Canada exports approximately $4.3 billion worth of seafood products each year. To protect this trade Canada must meet international standards set by organizations such as the World Organization for Animal Health (Office International des Epizooties, or OIE), which sets standards for controlling diseases of international trade importance.

Our research helps set international standards for diagnostic tests - including the development and validation of new molecular assays. The application of molecular tools also demonstrates that organisms once believed to be pathogens of international concern are in fact innocuous (benign) host-specific parasites. DFO research into the development of DNA vaccines and how fish respond to these treatments provides additional health management tools to the Canadian aquaculture industry. Enhancing health through vaccination and other husbandry activities minimizes any risk that cultured aquatic animals will serve as a source of infection for susceptible wild species.

The major advantage of molecular tools is their specificity and sensitivity as applied to understanding diseases, disease progressions, host /carriers, and opportunities for mitigation and prevention.

Goal:

By 2015, to have developed and applied leading edge biotechnology-based techniques to detect, monitor and minimize the impact of pathogens on aquatic animals and apply this information to assess and improve the health of aquatic animals.

Objectives:

1. Develop, validate and employ molecular techniques to detect and identify endemic and exotic pathogens.
2. Incorporate molecular techniques in studies on epidemiology and transmission of aquatic pathogens for disease management.
3. Apply biotechnology-based techniques for the treatment and prevention of aquatic animal diseases.
4. Integrate biotechnology and other technologies in assessing the impact of disease in aquatic animals through risk analysis.

Objective #1

Develop, validate and employ molecular techniques to detect and identify endemic and exotic pathogens.

Action #1:

Develop, validate and apply reliable gene-based tests for parasites and pathogens.

• The detection of aquatic animal pathogens remains a priority for DFO and the seafood sector as a whole. Molecular techniques provide sensitive and pathogen- specific tools to meet these obligations.
• Currently, validated molecular tests are not available for most pathogens of concern, and biotechnology is therefore under-utilised in aquatic animal health. There is a need to develop, validate and apply this technology to investigate aquatic animal diseases.
• The development and application of molecular techniques in Canada is necessary for the implementation of a national surveillance program. Surveillance is internationally required to demonstrate freedom from diseases of economic and ecological concern.
• Licences required for product export may be issued only if health certification is based on sensitive and specific diagnostic techniques. Certification confirms that the product is free of disease agents that may be harmful to the protection and conservation of fish, and therefore, facilitates trade and the activities of the aquaculture industry.

Action #2:

Identify and characterize emerging pathogens of economic and ecological concern.

• As aquaculture techniques are developed for commercially valuable native species, previously unknown diseases are likely to be encountered. Identifying the disease agents and understanding their biology and pathogenesis will be facilitated by the application of biotechnology.
• The application of molecular techniques will significantly decrease the time required to identify emerging pathogens, to evaluate their dispersal in the environment and to determine the disease status of different geographical zones. This information will contribute to the prevention and/or control of emerging pathogens economic and ecological concern.

Did you know that…

DFO scientists identified a "universal non-metazoan" polymerase chain reaction assay that selectively amplifies a segment of the non-metazoan Small Subunit rDNA gene. This assay was validated as a powerful tool for obtaining molecular information on pathogens that have not been isolated from metazoan host tissue. Thus, solving the dilemma of identifying the DNA of protistan pathogens that cannot be obtained free of host DNA, which is usually amplified by the application of conventional universal primers.

Action #3:

Establish standard quality assurance and quality control methods and take steps to facilitate their general use.

• Successful application of molecular diagnostic techniques depends on the reliability of the results. Strict applications of prescribed procedures in a quality assurance and quality control (QA/QC) laboratory setting will ensure this reliability.
• Validation of novel molecular techniques will assure that the assays can be reliably reproduced in all certified QA/QC laboratories.
• The strength of our seafood exports will be enhanced if Canada maintains the level of expertise that is required to meet the ever changing international standards that reflect the emergence of new technologies of disease control and surveillance.

Did you know that…

DFO scientists use polymerase chain reaction-based (PCR) test to differentiate between MSX and SSO infections in oysters during the outbreak in Nova Scotia. The differentiation allowed the control measures to be concentrated on areas affected by the more pathogenic MSX infections and limited the economic impacts of culture operation closures. As a result of the Canadian diagnostic experience, the Office international des epizooties (OIE) has declared the PCR confirmation as the international standard for the diagnosis of MSX and SSO infections in oysters. Canada, through its extensive research, has gained international recognition as a world leader in molecular diagnostic techniques.

Objective #2

Incorporate molecular techniques in studies on epidemiology and transmission of aquatic pathogens for disease management.

Action #1:

Use high resolution genetic typing techniques to characterize economically significant pathogens.

• To enhance our understanding of pathogenesis, the development and application of molecular tools to detect pathogens in situ, and thus characterize pathogen dissemination within and between hosts is needed.
• Understanding the genetic types or strains of a particular aquatic pathogen that exists throughout geographic zones over extended periods of time is essential in determining epidemiological inferences.
• The characterization of specific strains of disease agents supports the implementation of appropriate responses (e.g., virulent vs. avirulent strains of some viruses require different control measures).
• The establishment of epidemiologically-robust molecular-based pathogen surveys of wild and cultured stocks of aquatic animals will serve to identify trends in the health of these populations and to identify new potentially harmful pathogens. The detection of sub-clinically infected animals allows for the application of preventative control measures to limit disease outbreaks.
• Genetic typing, combined with phylogenetic analyses, provides information on pathogen evolution, which can be used to develop sensitive molecular assays that can be applied to understanding pathogen dispersal and mode of transmission. The ability to track pathogens that undergo differentiation and transmission through unexpected routes, such as host reproductive products or intermediate host(s), will allow for the application of measures to prevent disease transmission.

Action #2:

Develop and implement an accessible aquatic pathogen genetic strain database.

• A database documenting the genetic variability among strains of a particular disease agent will facilitate fish health management decisions on the movement of infected fish stocks as well as provide epidemiological information detailing transmission events and disease sources.
• Information on the expression of genes within a host exposed to selected pathogens, generated through the use of microarrays, add to our understanding of the genetic and physiological impact of the pathogen on the host. Through the collection and comparison of this type of data, additional information on the similarities and differences in host-pathogen responses and the impact on gene regulation can be generated.

Objective #3

Apply biotechnology-based techniques for the treatment and prevention of aquatic animal diseases.

Action #1:

Develop biotechnology-based therapies for aquaculture and hatchery aquatic animals.

• Infectious diseases are a major constraint to the development and success of aquaculture in Canada and negatively impact stock enhancement programs. The development of prophylaxes against economically significant pathogens will greatly benefit the survival of aquatic animals in culture facilities. Biotechnology and genomics tools can be applied to the development of new therapies against aquatic pathogens to not only benefit aquaculture, but also reduce the spread of pathogens to wild stocks. In addition, using molecular techniques to develop and evaluate fish responses to new therapies against aquatic pathogens of concern will speed up the development phase of these prophylaxis treatments.
• DNA vaccines are proven to be effective against some virus pathogens of finfish and overcome many of the problems encountered with conventional vaccines such as safety, cost and storage. Considerable basic genomics research is still required to identify the appropriate genetic elements of most known parasitic, bacterial and viral pathogens.
• Conventional vaccination of cultured finfish involves intraperitoneal injection or immersion delivery, whereas current technology requires the intramuscular delivery of DNA vaccines. Research is necessary to explore alternative delivery methods for DNA vaccines and ideally, to integrate the delivery of conventional and DNA vaccines.
• DNA vaccines and other new therapies such as heat shock protein-based recombinant vaccines will be explored as new therapies for hatchery reared animals.

Action #2:

Use biotechnology to understand the host immune response to natural infections and follow vaccination against specific pathogens.

• The presence of infection is a necessary, but not unique prerequisite for disease. The ability of an aquatic animal to defend itself against the effects of infection will determine the outcome of infection. Basic research is required to better understand how aquatic animals respond to infection and to vaccination. Biotechnology can provide molecular markers of exposure and fish response to infection. Knowledge of the defense mechanisms of aquatic animals will enable the development of "host response" molecular diagnostic techniques. The combined ability to diagnose infection and host response to infection will permit a more thorough assessment of the health of wild and cultured species.
• Significant progress has been made elucidating many of the genes relevant to the salmonid immune system and these gene sequences provide new tools for studying the teleost immune system response to pathogens and vaccines.
• By collaborating nationally and internationally through the Genome Canada projects on aquatic organisms such as the Genomics Research on Atlantic Salmon Project (GRASP) or the Consortium for Genomics Research on all Salmonid Projects, and by using technologies such as qRT-PCR and microarray analysis, DFO laboratories will investigate expression changes of important cytokine and immune response genes during natural infections or following immune stimulation due to vaccination. New markers may also be identified and differential responses to pathogen variants are expected.

Objective #4

Integrate biotechnology and other technologies in assessing the impact of disease in aquatic animals by risk analysis.

Action #1:

Work closely with CFIA to develop a formal risk-analysis process for established and emerging pathogens and diseases of aquatic animals.

• Biotechnology will provide pathogen and host response data of sufficient resolution that together with conventional tools, will substantiate the foundation of risk analysis. The risk analysis approach may be useful in establishing priority pathogen/host/disease combinations for policy development or research.

Theme #3: Biotechnology and Aquatic Ecosystem Health

DFO's mandate is to conserve, protect and enhance aquatic ecosystem health. Healthy and productive aquatic ecosystems are not only home to an enormous number of species, but also the basis for a thriving resource industry. Effective conservation and protection of this valuable resource remains a challenge with so much to learn about the living organisms in aquatic environments, their life-cycles and broader ecosystem structure and functions.

While we are a long way from fully understanding ecosystem dynamics, recent advances in biotechnology enable us to assess and in certain cases, mitigate the impact of anthropogenic and environmental stressors. For example, changes in community structure and function can be monitored using new techniques in meta-genomics and novel bioremediation techniques such as biostimulation, and bioaugmentation can be used to treat contaminated sites.

DFO has a history of monitoring contaminated sites in aquatic environments. With increased concern over the negative impact of contaminants on the ecosystem including fish habitat and human health, the Department takes a proactive approach to site remediation. In this regard, habitat restoration is now a recognized component of the Oceans Action Plan.

Healthy ecosystems are the basis for biodiversity, healthy communities and development. Environmental health assessments are an essential component of integrated management initiatives including protection, conservation, mitigation and/or restoration. Biotechnology and genomics tools can generate information about populations, individuals, physiological and metabolic responses to alterations, all of which can provide discrete information that can be integrated into models and approaches for evaluating ecosystem integrity.

Goal:

By 2015, to develop and apply biotechnology and genomics tools to enable assessment, mitigation and restoration of aquatic ecosystems.

Objectives:

1. Develop and apply genomic indicators to detect and monitor environmental stress in aquatic ecosystems.
2. Develop genomic tools to understand biological processes for mediating natural recovery in contaminated sites, and for development of bio-remediation technologies for mitigation.
3. Develop sensitive tools based on genetic methods to detect and monitor invasive species and assess potential impacts.
4. Improve measures of ecosystem health using meta-genomics and other biotechnology and genomics tools.

Objective #1:

Develop and apply genomic indicators to detect and monitor environmental stress in aquatic ecosystems.

Action #1:

Evaluate biological stress indicators, using biotechnology and genomics tools, for key species and ecosystem components within various aquatic ecosystems

• Ecosystem components may respond different to the same environmental stress, resulting in the need to develop and implement tools that can detect environmental stress within different organisms representing different ecosystem components, including microbial populations and sensitive species.
• Changes in fitness and genetic alterations in fish can been used as indicators for environmental stress. For example, genetic markers have been identified that are tightly genetically linked to the sex determination locus in salmon, thereby allowing for unambiguous determination of genetic sex independent of the developmental state of the gonad. These genetic markers provide a valuable tool for sensitizing assays identifying endocrine disruption effects, like gonadal sex reversal, which have been linked to exposure to effluents in the environment. Evaluation and further development of sensitive tools like these allow researchers to quickly and sensitively monitor the effects of changes in the environment and provides valuable information for evaluating ecosystem integrity, and making recommendations for changes in human-based activities.

Action #2:

Develop and apply biotechnology and genomics tools to to detect environmental alterations.

• The application of genomics to identify changes in the genetic structure of living organisms and the way in which they function provides a unique tool for the delineation of impacted zones and as a means to monitor contaminant degradation potential and/or habitat recovery.
• Gene expression profiling distinguishes altered physiological pathways in fish in response to environmental changes, and may provide an "early warning" system for detecting pollution, stress, habitat degradation, etc., in the aquatic environment.
• Genomics capability allows for the investigation into the effects of alterations in the environment on the physiological, metabolic, protein and gene expression within aquatic organisms. From this information, potential end point indicators can be identified through the evaluation of reference sites or laboratory studies that focus on the impact of habitat degradation, pollution, climate change, etc on the organism.

Objective #2

Develop genomic tools to understand biological processes mediating natural recovery in contaminated sites, and further develop bio-remediation technologies for mitigation.

Action #1:

Develop genomic tools to characterize biological processes that remediate contaminated sites.

• Develop genomic assays to identify, isolate and characterize microorganisms responsible for the biodegradation and biotransformation of contaminants.

Action #2:

Develop biostimulation and bioaugmentation methods to promote the biodegradation and/or biotransformation of contaminants.

• Laboratory development and evaluation of potential bioremediation strategies for use in contaminated aquatic environments.
• Develop application methods for bioremediation strategies and protocols for monitoring treatment efficacy.
• Assess bioremediation strategies in field trials and publish guidance documents for technology transfer to internal (resource managers) and external (industry) clients.

Did You Know That…

The National Centre for Offshore Oil, Gas and Energy Research is developing new sensitive, cost-effective and rapid assays, based on recent advances in biotechnology for monitoring habitat recovery. A coupled application of analysis in meta-genomics and physical oceanography has improved our understanding of natural recovery and the feasibility of pro-active remediation procedures in contaminated harbours (e.g., Sydney Harbour). Bioremediation strategies developed by DFO have provided direct benefit to government emergency response agencies (e.g., Canadian Coast Guard) and private sector industries that offer advice and products for oil spill cleanup on a national and international scale.

Objective #3

Develop sensitive tools using genetic methods to detect and monitor invasive species and assess potential impacts.

Action #1:

Develop tools for early detection of invasive aquatic species.

• Invasive species cost the Canadian economy billions of dollars annually and wreak havoc on aquatic ecosystems. There is international recognition that invasive species are a global environmental, economic and political issue warranting urgent attention.

Action #2:

Develop tools to assess and mitigate pathogens and parasites associated with exotic species.

• Ballast water may contain exotic species, and associated pathogens and parasites, which may be difficult and time-consuming to identify using conventional techniques. Through biotechnology and genomics tools and information scientists can quickly and accurately assess which pathogens, parasites and exotic species are found in a sample.

Objective #4

Improve measures of ecosystem health using meta-genomics and other biotechnology and genomics tools

Action #1:

Examine microorganism gene pools within aquatic ecosystems to monitor degradation or recovery (metagenomics).

• Microorganisms are essential for the basic metabolic processes controlling ecosystem function such as primary production, nutrient cycling, the biodegradation and biotransformation of contaminants. Advances in biotechnology and genomics will help us better understand their community structure and function.
• Studies on the application of meta-genomics (i.e., quantification of changes in genomic structure and expression in natural microbial populations as a means to identify changes in environmental conditions and/or ecosystem health), have been conducted in partnership with the Biotechnology Research Institute (BRI) of the National Research Council located in Montreal, Quebec.
• Metagenomics could be applied to study the impact of seawater netpens on the benthic microbial community. A progression of microbial populations within these communities has been observed, and metagenomics could be applied to track this progression and the rate and effect of changes in these microbial communities in response to the presence of aquaculture activities.
• Metagenomics could also be applied to identify the microbial communities that are currently found frozen in northern arctic sea-ice. This could help researchers understand whether these bacteria are still viable, whether there are pathogens present in these communities, and thus contribute to the knowledge and understanding of the historic microbial community within the arctic.

Action #2:

Generate ecosystem integrity information, using biotechnology and genomics tools that can be integrated into aquatic ecosystem science management approaches.

• Biotechnology and genomics tools can provide information that helps to identify changes in biodiversity, and in some cases, can reveal linkages between ecosystem health and the health and biodiversity of species.
• Linkages between biodiversity changes and ecosystem integrity can be very complex, and although the information generated through biotechnology and genomics tools can greatly enhance our understanding, ecosystem science and ecosystem management approaches will continue to require additional biological and ecosystem science information as well as refinement. Ultimately, the aim is to be able to detect and interpret linkages, which can contribute to informing management decisions, setting ecosystem objectives, and supporting actions to restore or enhance ecosystem health.

Action #3:

Develop and apply the expertise to enable the evaluation of the biological significance and compatibility of genomics, proteomics and metabolic profiling data, and the integration of this data into ecosystem integrity models.

• As genomics, proteomics and metabolic profiling tools and techniques become more sophisticated and are increasingly integrated into research, the quantity of data that is produced rapidly increases. A critical function that will have to be addressed will be both the standardization, evaluation and handling of data from specific, related experiments, as well as addressing questions on how to evaluate and integrate information from multiple sources and experiments.
• Biotechnology and genomics tools can provide information on changes in environments (e.g., population structure), and responses to environmental changes (e.g., gene expression changes), but do not immediately indicate that an impact has occurred. In order to asses impacts, the genomics information will need to be integrated into existing in-house DFO knowledge and understanding of ecosystems and aquatic organism biology, and placed in context of the normal variance seen for ecosystems and organisms.
• Through the further development, understanding and application of biotechnology tools, more refined estimates and interpretations of ecosystem impacts, habitat recovery and overall ecosystem integrity can be incorporated into scientific advice given to support DFO's activities to conserve, protect and enhance aquatic ecosystems.

Theme #4: Regulatory Science for Aquatic Animals with Novel Traits

DFO is responsible for the regulation of aquatic organisms with novel traits under the New Substances Notification Regulations (Organisms). In support of this regulatory responsibility, DFO undertakes a research program which involves the development and assessment of aquatic animals with novel traits, including transgenic fish. The majority of this research takes place in the Centre for Aquaculture and Environmental Research (CAER) in British Columbia, with other projects at the Pacific Biological Station in Nanaimo, British Columbia, and Bedford Institute of Oceanography in Halifax, Nova Scotia.

Included in the scope of organisms addressed under this theme are aquatic animals that express a trait (or traits) that is new to the organism, is no longer expressed in the organism, or is expressed outside the normal range of expression for that trait in that organism. In order to obtain factual information on performance characteristics, fitness parameters and food safety characteristics of aquatic animals with novel traits, DFO has developed non-commercial salmon strains with novel traits using conventional approaches such as selective breeding and modern biotechnology. This information is important in order to assess potential impacts that escaped fish with novel traits might have on wild populations. The transgenic strains are also used by other federal regulatory departments and agencies (e.g. Health Canada and the Canadian Food Inspection Agency) in support of biotechnology regulatory responsibilities.

Our program has identified regulatory science issues that must be addressed in the design of DFO's regulations including the interactions with wild fish; data requirement hurdles such as sample size uncertainty; lab scale uncertainty; the scope of "novelty"; the effectiveness of containment approaches; and what information is needed in order to complete a risk assessment.

Goal:

By 2015, to undertake research to provide sufficient understanding to be able to assess the use of aquatic novel living organisms and to allow effective regulation.

Objectives:

1. Enable risk assessment science through the identification, development and evaluation of appropriate novel aquatic animal models.
2. Conduct studies in support of risk assessment methodology and the design and implementation of regulations.
3. Develop and evaluate the efficacy of preventative and mitigative measures to prevent interaction between wild and novel aquatic animal strains (containment strategies).
4. Assess potential ecosystem impacts of transgenic aquatic animals.

Objective #1:

Enable risk assessment science through the identification, development and evaluation of appropriate novel aquatic animal models.

Action #1:

Identify domesticated and invasive aquatic animal species and strains with potential threats to Canadian ecosystems.

• Aquatic animal strains that differ phenotypically or genetically from those found in a specific ecosystem being considered have the potential to have short or long term effects on conspecifics and other members of the ecosystem. Examples include strains of Canadian aquaculture species that have been selectively bred to possess characteristics not found in nature; fish which have been enhanced in mass scale in hatcheries; foreign introduced species; and invasive "stowaway" species.

Action #2:

Develop and maintain contained transgenic strains of aquatic animals to inform regulatory development.

• It is difficult to evaluate novel aquatic animals through modelling and theoretical assessments due to the complex unpredictability of phenotypes arising from many genetic modifications. Thus, assessments are best performed on actual transgenic organisms. In Canada transgenic aquatic animals are being developed for commercial use, but such animals are not available for risk assessment research by the federal government.
• The synthesis of similar public domain strains for use by DFO, other government departments, and other national and international researchers is critical to allow development of empirical risk assessment information in support of emerging regulations. Information derived from this collaborative and public domain approach greatly enhances the knowledge base for DFO scientists and regulators on transgenic organisms, keeps DFO in contact with the latest developments in the field and creates access to a network of collaborative expertise.

Action #3:

Evaluate environmental parameters required for growth, survival and overwintering of common R&D aquatic animals, particularly those that are not native to Canada

• Aquatic animals are increasingly being used as models for research and development purposes, due to the rapid growth and short life-span for common R&D aquatic organisms like zebrafish and medaka. The generation of knowledge of the ranges and tolerances to environmental parameters required for growth, survival and overwintering in Canada will provide regulators with scientific information that can be incorporated into the design and implementation of regulatory approaches for the oversight of R&D activities involving novel aquatic animals.

Objective #2:

To conduct studies in support of risk assessment methodology development and the design and implementation of regulations for novel aquatic organisms.

Action #1:

Evaluate specific phenotypes of various strains of novel and domesticated aquatic animals in order to better determine key parameters that influence environmental risk.

• Physiology provides the link between genotype and phenotype and behaviour, and thus can provide a critical insight into processes that are affected by transgenesis and domestication. Experiments are needed to measure key survival fitness characteristics (such as risk taking behaviour, growth and energy metabolism, reproductive performance) and reproductive fitness characteristics (spawning success, fertility, fecundity, gamete quality) of transgenic and domesticated salmon. The results of these studies will provide knowledge to help in the assessment of possible risks to wild salmon should the transgenic salmon escape to natural environments, and to identify key parameters to investigate in newly developed aquatic animals.
• Assessments of behaviour, physiology and genetics of wild, domesticated and transgenic strains provide baseline data and knowledge that are used to develop the optimum approaches needed for risk assessments.
• Similar phenotypes can arise through the application of selective breeding or modern biotechnology, including recombinant DNA techniques. Detailed comparisons of domesticated and transgenic strains are required to determine the nature of genotypic changes resulting from traditional selective breeding techniques or recombinant DNA techniques, respectively. Empirical studies of genotype/phenotype relationships can help provide clarity for the definition of a regulatory trigger.

Action #2:

Evaluate ecosystem impacts and fitness of transgenic aquatic animals using model systems, prior to their release.

• The main objectives of risk assessments of transgenic fish are: 1) to evaluate the phenotype of the animal compared to nontransgenic animals to estimate the potential impacts the former may cause on ecosystems; and 2) evaluate the relative fitness of transgenic organisms to determine if they can effectively compete in nature and persist through subsequent generations. To undertake these assessments, knowledge of factors affecting the ability of the novel organisms to survive to maturation and their ability to reproduce are critical to assess the overall lifetime fitness.
• Many factors continue to be evaluated to assess survival and reproductive performance, invasiveness, and potential ecosystem effects (e.g., resource utilization or habitat degradation), using simple laboratory apparatus (e.g., swim tunnels, disease challenge facilities, behaviour chambers, nutrition tanks, etc.)
• Comparisons of behaviour (predation avoidance, competitive growth, spawning success) are underway for nontransgenic and transgenic fish either raised in tanks or semi-natural environments. However, there is much uncertainty in the conduct of empirical research of transgenic aquatic animals because the way an organism interacts with its environment profoundly affects survival and reproductive fitness. While such information provides the foundation for planning more detailed investigations, more complex experimental habitats are urgently required to undertake such studies under conditions that simulate natural conditions more realistically.
• Information from the semi-natural environments developed to date indicates that much more complicated interactions among fish of different genotypes occur, and that pleiotropic phenotypic traits are extremely complicated to evaluate due to genotype by environment interactions, because different data is generated depending on the experimental conditions employed. These observations suggest that risk assessments based on data generated in simple laboratory apparatus may have very little practical use for assessments of risk to the natural environment.
• Pleiotrophic effects in fish are known to be significant. Therefore, the process by which a novel aquatic animal has been developed may influence other traits and phenotypic expression, particularly when the method uses modern biotechnology. In order to design and implement appropriate and effective regulations, information on the effect of the method (or process) used to develop the novel aquatic animal is needed.

Objective #3:

Develop and evaluate the efficacy of preventative and mitigative measures to prevent interaction between wild and novel strains (containment strategies).

Action #1:

Develop and evaluate biological containment methods.

• Biological containment options for finfish and shellfish have been identified and developed as methods to limit the impact of these novel aquatic animals on aquatic ecosystems, by preventing interbreeding with wild strains. The evaluation of the efficacy of biological containment is required to determine its appropriateness as a containment method.
• Triploidy (pressure shock induced within all-female strains) is currently the most achievable method of inducing sterility in finfish and shellfish on a large scale. DFO now has a large series of experiments underway to test the efficacy of this approach for containment of finfish.
• Previous DFO research has shown that triploidy may not provide sufficient containment for some situations (e.g. use of transgenic strains). Thus, biological containment strategies including those involving transgenic techniques, that provide a higher level of containment are desirable, but have not yet been fully developed. These techniques require further development and efficacy assessment which may in turn require the development of model strains.

Action #2:

Evaluate physical containment methods for tetraploid shellfish broodstock.

• Physical containment strategies are required for preventing environmental release and interactions with wild populations of fertile tetraploid shellfish broodstock that are used to produce triploid shellfish. Potential challenges for physical containment exist due to the lifecycle and lifestages of shellfish. Therefore, scientists need to evaluate the efficacy of various physical containment methods in order to inform the development of appropriate standards and approaches for effectively containing these fertile aquatic animals.

Action #3:

Evaluate mitigative strategies for limiting and/or preventing interactions between wild and novel aquatic animals.

• The development and evaluation of mitigative strategies is important as an backup approach to preventing interactions between wild and novel aquatic animals. One such approach is to develop conditional expression systems that can control survival and reproduction of escaped individuals in nature.

Objecitive #4:

Assess potential ecosystem impacts of transgenic aquatic animals

Action #1:

Generate knowledge to enable the evaluation of potential ecosystem impacts resulting from intentional mass introductions of novel aquatic animals into various environments

• Potential ecosystem impacts from mass introductions (e.g., triploid shellfish) will depend, among other factors, on the type of introduced novel aquatic animal, it's novel trait, the ecosystem it is introduced into, including the presence of conspecifics or competitors, the lifecycle of the introduced organism, adjacent ecosystems, and the current environmental conditions. These factors and their interactions will require an ecosystem approach, integrating information generated from various studies.
• Determination of potential ecosystem impacts is necessary for evaluation of likely short and long term impacts resulting from mass introductions of novel aquatic organisms both on the ecosystem into which it is intended to be introduced, and adjacent ecosystems.

Action #2:

Evaluate potential ecosystem impacts resulting from unintentional releases of novel aquatic animals into various environments

• The potential ecosystem impacts that may result from an unintentional release of novel aquatic animals, particularly novel fertile aquatic animals, will need to be evaluated and the information generated from these studies incorporated into regulatory considerations. Potential impacts of an unintentional release of novel aquatic animals will likely be influenced by the scale of the unintentional release. The development and evaluation of models that take into account the scale of unintentional release in addition to factors that would be used to estimate potential ecosystem impacts of an intentional introduction, can inform estimates of potential ecosystem impacts from unintentional releases.

Action #3:

Evaluate ecosystem factors that may influence competitive ability of novel aquatic animals.

• Ecosystem factors, such as the size and complexity of a habitat, the type and availability of food resources, population density and the potential for migration within the ecosystem, are thought to influence the competitive ability of aquatic animals. By evaluating the significance and complexity of such ecosystem factors, scientists will be able to identify which factors are likely to effect predictions of behaviour and survival of novel aquatic animals in an ecosystem, which are important for risk assessment models.

Action #4:

Generate knowledge to better understand the nature of ecosystems that may be most affected by novel aquatic animals

• The generation of baseline data (e.g., species diversity, food resources, and habitat availability) on the different ecosystems that may be impacted, particularly from the introduction of anadromous novel aquatic animals (e.g., salmon), is required to further develop the knowledge base upon which assessments can be made.