AVCLSC Update February 2004

AVC Lobster Science Centre - linked to home page

Volume 2, Issue 1

February 2004
Quarterly Update from AVCLSC
2003 ANNUAL REPORT SUMMARY
In this Issue:

A quarterly communication from the AVC Lobster Science Centre at the
University of Prince Edward Island in Charlottetown, PEI, Canada

A following is a summary of the
2003 Annual Report (January to December 2003)
of the AVC Lobster Science Centre.
The full report is available upon request - see contact information at the end of this report.

Director's Message
Infectious Disease & Host Response:
Immunity , Proteomics , Peptide
Paramoebiasis
LobsterNET
Finances
Chairman's Remarks

Scheduled to begin in January 2004, some initial work was carried out in January-March 2003 due to the availability of personnel and equipment. Hemocytes, invertebrate equivalents of mammalian white blood cells, comprise a major component of the innate immune system. Special hemolymph (lobster blood) collection and handling techniques are essential for performing hemocyte function assays. Different anticoagulants and sample preparation techniques were being evaluated to determine which will provide optimal hemocyte stability. A manuscript entitled “Classification of Homarus americanus hemocytes and the use of differential hemocyte counts in lobsters infected with Aerococcus viridans var. homari (Gaffkemia)” has been published in the Journal of Invertebrate Pathology. This paper presents a new classification scheme for lobster hemocytes, currently used at the AVCLSC, which will allow researchers to use differential hemocyte counts in lobsters to obtain information similar to that obtained from differential white blood cell counts in veterinary and human medicine.

To productively investigate the innate immunity of lobster requires attention to aquarium conditions and animal handling that exceed those followed for most other shellfish. The natural habitat of the lobster is pristine, and it is only because of a remarkable immune system that the animal is able to tolerate live-market conditions. We have to study the lobster immune system under pristine conditions, as it would exist in the wild, to understand how it can deal with the conditions of live-holding. This required an adjustment to the physical facilities and how experimental animals would have to be conditioned and maintained. The problem of having to schedule animal experiments according to the local fishing seasons, and the problem of having no measure of the post-capture stress for these experimental animals, compromised our ability to be efficient with our experiment scheduling, or be assured of the validity of our results. A conditioning period for the wild-caught lobsters, in which there is an acclimation to a new environment and a formulated diet, gives us greater confidence in our results and frees us from the confines of fishing seasons. We have to study the immune system’s response to the sorts of infectious agents that lobsters have to successfully deal with under conditions of live-holding. Aerococcus viridans, the bacterium causing Gaffkemia, is an infectious agent that lobsters do not successfully deal with. Hyphomicrobium indicum, the bacterium associated with “Limp Lobster Syndrome”, is one of the infectious agents that the lobster occasionally does not successfully deal with. The experimental model for Gaffkemia has successfully been developed; the model for “Vibriosis” has not. Development of this “Vibriosis” model is now being integrated into the plan. Incorporation of these pathogen models is essential for completing an assessment of lobster innate immunity. The disease model approach to studying humoral immunity is conservative but classic: the understanding of human humoral immunity began with the disease model approach. The concept is straightforward: those elements of hemolymph that bind to the bacterium Hyphomicrobium indicum have the potential to be elements of a successful defence reaction. There are alternative approaches to this research now available to us, and Sara Purcell MacDonald, an AVCLSC-funded PhD candidate, is approaching the same humoral immunity problem with one such alternative. She is looking for lobster DNA that codes for cell-level receptors for one of those elements that would bind to the bacterium. Sara’s work compliments the approach taken for the study of cellular immunity as well, given that the hemocyte surface is the expected location of the receptors.

With the unforeseen delays in the Paramoebiasis project, the proteomics activity on the Gaffkemia agent Aerococcus viridans var. homari was moved ahead. Appropriate two-dimensional electrophoresis equipment and analytical software were purchased. A major boost to the project was the acquisition of a significant collection of bacterial isolates (donated by Dr JE Stewart, DFO). This collection included both known virulent and avirulent Aerococcus isolates from lobster, as well as commensal bacteria isolated from the gut of lobsters. This collection, in conjunction with bacterial isolates from the American Type Culture Collection and our AVCLSC case isolates, has provided an excellent bank of material for further characterisation. There is minimal published information on the genetic similarities and differences between defined Aerococcus isolates. However, having a solid genetic and biochemical background of the specific isolates is a required prerequisite to pursuing the proteomics. We have taken considerable time in defining the genotypic and phenotypic differences between the isolates. This characterisation has provided us with an appropriate and sufficient foundation in which to incorporate solid testable hypotheses into our analysis of the proteome of Aerococcus viridans var. homari.

One of the overall objectives of the research activity is to identify pathogenic features of microorganisms causing disease in lobsters. To fully capitalise on the investment in our proteomics technology, we want to explore in parallel, other pathogens of economic importance to the lobster industry. However, there is often a limitation to proteomics projects in that they require the availability of genomic sequence data to correctly identify proteins. This is especially a concern for eukaryotic organisms like protozoa where genome size is increased (relative to the average bacteria) and there are fewer genomes for comparison. In this way, genomics research complements and extends proteomics research by providing the gene database from which the rapid identification of proteins can be determined. We intend to pursue genomics research on Anophryoides haemophila, the ciliated protozoan that causes “Bumper Car” disease in lobsters. The rationale is that we can construct and then exploit, through proteomics, our own annotated genomic database. From previous research on “Bumper Car” disease, the AVCLSC has developed expertise that has been published in scientific journals and the information incorporated into management strategies within the lobster industry. Therefore, a “Bumper Car” genomics project would extend our knowledge of this disease by applying the latest scientific technologies and begin to provide us with a clearer picture of how this parasite causes disease in lobsters. The opportunity has arisen to collaborate with the ‘Protist EST Program’ (PEP) at Dalhousie University. The PEP group includes eight other researchers from across Canada and is funded through Genome Atlantic. The aim of their program is to compare the genomes from many protists (protozoa) to discover information about their protein-coding genes (EST or expressed sequence tags). These new proteins may have novel properties and activities that could have medical and scientific benefits, or have commercial applications. The PEP group is looking to expand their protozoan genomic database for comparative analysis by establishing collaborations with researchers with similar interests within Atlantic Canada. The AVCLSC was a logical choice for collaborators, with recognized expertise on protozoan parasites of crustaceans and on protozoan molecular biology. The primary benefit of this collaboration to the AVCLSC is that we would have access to the PEP group’s collective expertise in EST sequencing analysis and their computer analysis software that allows for highly rapid gene identification and annotation. The AVCLSC, by providing all the funding through the PEI Atlantic Shrimp Corp Inc, will maintain all intellectual property derived from the “Bumper Car” genomics project. Timely completion of the “Bumper Car” genomics project would allow the AVCLSC to make the transition more quickly to the proposed second phase of the project, “Bumper Car” proteomics.

Completion of the task (to isolate, identify, characterise & localise antimicrobial peptides [AP] in lobster blood cells & plasma) requires the integration of multiple techniques and procedures. The method for preparing the hemocyte lysate supernatant (HLS) has been modified to increase sample recovery. Equipment was purchased to perform polyacrylamide gel electrophoresis (PAGE) a technique used for determining the size (molecular weight) of the peptides. Chromatography equipment has also been purchased. After the antimicrobial peptide(s) has been identified, isolated, and purified, it will be submitted to a specialised laboratory for amino acid sequence analysis. The results will be used to compare the lobster antimicrobial peptide to known peptides in other species. A semi-quantitative ‘spot assay’ has been established and is in use using two test bacteria, in-house isolates from lobster intestine. Testing of HLS has confirmed the presence of antimicrobial activity, likely due to the presence of one or more antimicrobial peptides. Completion will require testing of the purified peptides. As work continues, we intend to increase the spectrum of test bacteria used in the assay and establish protocols for a quantitative micro titre plate assay.

Our initial approach is to obtain material from CCAP because this group ships live cultures of amoebae; in contrast, the ATCC ships frozen material which can be challenging to establish in vitro. Neoparamoeba pemaquidensis is the causative agent of mass mortalities in sea urchins and amoebic gill disease of finfish. Having ready access to multiple sources of the amoebae should increase our success in transmitting the parasite to lobsters, and increase our understanding of the parasite and its interactions with its hosts. The parasites previously known as ‘Paramoeba’ spp. have now been more correctly classified as members of the genus Neoparamoeba. The classification of the individual species within the genus has, however, not been resolved. These species have been presumptively grouped under the name Neoparamoeba pemaquidensis, which is considered a “species-complex” with multiple strains having different and varying life history strategies. The realization that N. pemaquidensis is a species-complex has “clouded” the issue of a specific pathogen causing a specific disease in a specific host. We hope to elucidate this issue through our combined alternative strategies (see below) to studying this disease process in lobsters.

A shipment of cultures of N. pemaquidensis from CCAP is thriving in its initial subpassages at the AVCLSC. We have now established a collaboration with the Bigelow Laboratory of Ocean Sciences (Maine, USA) which will provide us with cultures of N. pemaquidensis derived from sea urchins. Paramoebiasis of sea urchins can cause major epidemics throughout Atlantic waters; sea urchins are easy to access and maintain in the laboratory and may be an significant reservoir for the parasite. We intend to establish “infection tanks”, using either Atlantic salmon or sea urchins as hosts for N. pemaquidensis to ensure infectivity of the amoebae for in vivo experimentation. An alternate strategy, complementing access to in vitro cultures of amoebae, is to study Amoebic Gill Disease (AGD) of salmonids as a model to examine paramoebiasis in lobsters. Amoebic Gill Disease is a major parasitic disease of aquaculture operations with salmonids, turbot, sea bream and sea bass. We anticipate that recovering amoebae from gill tissues of salmon will be much easier than from lobster or sea urchin neural tissues. Our transmission experiments to lobsters will utilize conditioned lobsters on a feeding regime in full strength seawater at warm (10 ^oC or more) temperatures.

The N. pemaquidensis agent associated with the Long Island Sound (LIS) outbreak is intimately associated with the lobster neural tissue, which has made it impossible to isolate and difficult to detect using classical pathology techniques. Our goal is to be able to accurately assess the possibility of cross-species transmission of N. pemaquidensis among sea urchins, Atlantic salmon and lobsters and to be able to detect reservoirs of this pathogen from the environment. The development of diagnostics for monitoring and surveillance programs to assess the health of lobsters imported into Canada and to monitor the health of lobsters within Canadian waters will be developed. Paramoebiasis continues to be an important disease to study because of the spectacular collapse and ongoing low level of harvest of the Long Island Sound lobster fishery. The relationship of the disease paramoebiasis to this economic and ecological catastrophe has not been fully evaluated. The ongoing importation of lobsters from US waters into Canada for processing is a major reason to develop monitoring and surveillance methodology and programs for pathogens, including N. pemaquidensis. Additionally, global warming may be an important climatic factor in the potential movement of lobster pathogens northward into Canadian waters.

The overall objectives of Lobster^NET are: to allow processors to obtain up-to-date information on inventories & products that will help them optimise their processing, production and marketing plans; to provide an information service to the lobster processing industry by providing the benefits that other commodities have realised by adopting APHIN™ information technologies; to allow the Canadian scientists and the lobster industry to optimise research, processing, production and marketing decisions; and, to establish a ‘fee-for-service’ database service in Year 2 which will generate funds to be ‘reinvested’ into the AVCLSC’s annual budget. With these objectives in mind, the activity was divided into two Phases: the Construction phase scheduled to occur in Year One only; and, the Delivery Phase was scheduled to start in Year Two & continue throughout the remainder of this five year project. We are pursuing two significant research avenues with Lobster^NET in both Phases: the PEI Lobster Index Fishery & Sea Sampling Program; and the Central Lobster Access Website (CLAW).

We visited companies that showed interest in Lobster^NET and identified several data contributors for participating in Lobster^NET. This helped establish the specifications for the research database. With the involvement of the Maritime Lobster Processors Cooperative Ltd, presentations were made to specific industry players where an online prototype of CLAW was presented. Following a series of meetings, approximately two years of data on daily volume of lobster purchases from various buyers were obtained from industry partners in PEI and NS. This is significant as it represents the first datasets received from the industry. Substantial work has been done on the infrastructure of the website and on software programming and development for CLAW to deliver the information back to the industry. Additional meetings have been held with representatives of the industry to present the potential expansion of CLAW. We continue to receive industry datasets on a weekly basis, enabling us to continue our on-going work on the infrastructure of the site which is now available to the industry and represents the initiation of the Delivery Phase.

The PEI Index Fishery & Sea Sampling Program component of the DESIGN task is dependant on the starting date of the PEI Lobster Index Fishery and Sea Sampling Phase of the project and is awaiting appropriate permissions to proceed. We are designing a specific health-related database in 2004 for the CLAW project starting at the beginning of the 2^nd quarter of 2004.

As for the first task of the Construction component of the PEI Index Fishery & Sea Sampling project, this is also dependant on the actual starting date of the PEI Lobster Index Fishery and Sea Sampling project. This component will necessitate the development and testing of new software for the online information delivery using the back data. After this is completed and the current data added to the database, we will complete a pre-launch review of the program followed by the official launch and post-launch assessment. Presentations and meetings with industry and government representatives will also be necessary prior to the launch to ensure the success of the project. While we will continue to import current data from the CLAW project in 2004, we are planning the official program launch with a software review and a project review in the first quarter of 2004. Additionally, this component of the project will include the expansion of the data collected to include health data and more inventory data. We will need to develop new software for the website and further meetings and presentations from the Lobster^NET team will be necessary to obtain and refine the industry’s specifications, expectations, needs, requirements, and limitations for this expansion. For CLAW, it is still expected that in 2004, participants will be required to financially support the maintenance costs of Lobster^NET.

We have already started to discuss a fee-for-service system of payment with the industry, although more discussion will be necessary before a fair and equitable agreement is reached. It is estimated that the cost for the industry participant may be as low as pennies per pound.

Part of this task consists of maintaining, supporting, updating, upgrading and hosting the AVCLSC website. The AVCLSC website is the gateway to Lobster^NET and it has proven to be very useful via its internal team reporting features such as the team journals, notes and reports. Additionally, it serves as a vehicle for disseminating information and results generated by the AVCLSC to the general public, industry, other AVCLSC partners and collaborators and others. The next Task will consist of implementing the Lobster^NET system. We propose to include an increasing number of industry partners in the pilot team during the implementation component. This will allow us to assess the overall potential of the Internet-based information system. Data presently collected include product form (canners, markets) and volume purchased by buyer and by date. It is expected that additional data on inventories, products quality and health will be incorporated in 2004. As the databases become operational, a graduate student will use these data to conduct epidemiological and descriptive studies with sample sizes made possible only by this centralised, industry-wide data sharing system. The input to these databases will be on-going and evolving with new health parameters continuously added and monitored, enabling additional research to be performed.

The final task of this project will address the on-going evaluation and improvement of this lobster information system. Through continuous feedback from industry participants and provincial government representatives we will be able to enhance the program, ensuring a constant high level of quality.

The fiscal period for the project has been established as January to December for each calendar year, commencing January 2003. The first fiscal year (2003) had total costs of $1,884,514 (with $599,045 from the initiation period) and a contributed total financing of $2,570,707. Contributions for 2001-03 were received from: Acadian Fishermen's Co-op, ACOA, AVC Faculty Consultations, Canadian Gold Seafood, Clearwater Fine Foods Inc, Diagnostic Chemicals Ltd, Eastern Fishermen's Federation, ELANCO, Eskasoni First Nation, Exxon Mobil, Fisheries & Oceans Canada, Madelimer, Maritime Fishermen's Union, Maritime Lobster Processors Co-operative, Millbrook First Nation, Orion Seafood International, Paturel International, PEI Atlantic Shrimp Corp Inc, PEI Fishermen's Association, Polar Foods, Province of British Columbia, Province of New Brunswick, Province of Newfoundland & Labrador, Province of Nova Scotia, Province of Prince Edward Island, Royal Star, Seafood 2000 Ltd, Unama'ki Institute of Natural Resources, and UPEI Administration.

These meetings have occurred on wharves and meetings around kitchen tables. When the AVCLSC was envisioned, its central mandate has remained constant with the Centre’s mission being to ensure delivery of increased enhancement in sustainability of industry economic viability while respecting the need for resource health & biomass protection. As Chairman of the AVCLSC Management Council, I am extremely proud of the progress, growth, and knowledge acquisition that has been achieved to date. I am also impressed by the caliber and talents of the researchers and staff who have been recruited by the Centre. Their abilities, both individually and collectively, are resulting, and will continue to result, in knowledge that will enhance the value of the Canadian lobster industry to all stakeholders. While growth and enhancement is a never-ending task, the year has seen the placement of a strong foundation of capabilities which the AVCLSC partners can utilise to achieve the common goals of enhanced industry value and reinforced biomass health.

To what degree and at what speed we capture the opportunities which AVCLSC research can deliver will be a function of the level of support provided to the Centre. The AVCLSC is made up of partners in industry, in many levels and agencies of government, in First Nations, in academia, and in many science and technological collaborations. The unique environment created by the AVCLSC provides a vehicle for all stakeholders to work, shoulder to shoulder, to capture opportunities and to enhance the economic return from the Canadian lobster resource for the benefit of all Canadians.

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UPDATE is a quarterly communication from the AVC Lobster Science Centre at the University of Prince Edward Island in Charlottetown, PEI, Canada	Phone: (902) 894-2884 Fax: (902) 894-2885 Email: Lobster@upei.ca Website: www.LobsterScience.ca	AVC Lobster Science Centre University of Prince Edward Island 550 University Avenue, Charlottetown, PEI Canada C1A 4P3