Project Description
Project description:
Climate change is projected to cause elevated water temperatures with far-reaching effects on biochemical nutrient cycling, population ecology and shifts in species distributions. A crucial, though less appreciated, consequence of increasing water temperatures is the potential for severe disease outbreaks among wild as well as managed aquatic animal populations. Aquatic pathogens are likely to exhibit strong shifts in phenological, behavioural and life-history phenotypes in response to environmental change. However, the knock-on effects of enhanced transmission and virulence on host ecology, adaptation and evolution are poorly understood.
Proliferative kidney disease (PKD), caused by the myxozoan parasite, Tetracapsuloides bryosalmonae, is associated with freshwater salmonid fish species in the UK, Europe, and USA. Disease occurrence and severity are driven by increasing water temperatures with climate change trends aligning with increased disease outbreaks. Recent disease exacerbations have also been linked to an increasing diversity of susceptible fish hosts, such as whitefish populations in Europe and USA. Indeed, in the Yellowstone and Columbia River systems, recent major PKD outbreaks in farmed and/or wild trout populations have increased dramatically with increasing host ranges exemplified by mass die-offs of mountain whitefish populations reported in Yellowstone. Parasite strain diversity work in Yellowstone has revealed that moribund fish are infected by a distinct parasite strain, implying a genetic basis of parasite virulence in this system. PKD outbreaks also occur in Europe and has been observed in UK salmonid populations. Whilst no large UK mortality events have occurred to date, PKD still likely impacts the health of UK salmonid populations, and may, become a more serious threat as global climate change processes continue to evolve. A major barrier in tackling PKD is the lack of understanding of virulence and immune evasion. Studying PKD virulence and how it is affected by ecological and environmental factors in the USA, relative to the UK, will be highly informative for future disease management practices, especially as disease exacerbations appear to be driven by distinct strains.
Mechanisms by which parasites evade host immunity are often linked to disease pathogenesis (parasite virulence). The functional basis of immune evasion has been poorly characterised in most wild-life disease systems, despite its fundamental role in host-parasite adaptation and response to environmental change. This is mostly due to insufficient knowledge of genes directly contributing to immune evasion processes. A promising group of proteins implication in immune evasion are Intrinsically Disordered Proteins (IDPs) which play important roles in pathogenicity and are over-represented in pathogen genomes and have important roles in diseases, including COVID-19, malaria, and schistomiasis. For example, a group of IDPs called micro-exon genes (MEGs) encode host immune protein binding factors in helminth parasites and have been newly characterized in T. bryosalmonae.
We recently discovered a T. bryosalmonae fish specific secretory IDP, representing the first non-helminth MEG that acts as a putative virulence factor owing to its role in host immune evasion. TbMEG-1 functional studies have enabled visualization of parasites and development of serological tools validated on UK, European, and US trout serum samples. It exhibits alternative splicing resulting in numerous predicted protein isoforms capable of binding to a range of host proteins. Initial transcriptional surveys have indicated rich sequence diversity and isoform complexity in infected populations in UK, Europe, and USA. Distinct parasite strains are associated with moribund fish in Yellowstone. However, the range of TbMEG-1 sequence diversity and complexity is unknown among parasite strains and/or among hosts exhibiting different virulence levels. This project aims to investigate the relationships between environmental factors, pathogen diversity and host immunogenomic diversity at parasite IDPs implicated in host immune evasion in an emerging disease system of economic and ecological importance.
We will shortlist IDPs from the parasites genome and use targeted RNA-Seq to investigate links between IDP transcript repertoire / expression, parasite strain and virulence in whitefish and / or trout populations in the Columbia and Yellowstone rivers compared to those in UK rivers. The parasites will be genotyped using existing neutral markers to determine whether strain divergence and virulence phenotype are being driven by selection of distinct TbMEG-1 repertoires and other secretory IDPs enabling parasites to adapt to and exploit new host species. Fish populations will be sampled during the PKD season to determine how changing hydrological and temperature profiles influence diversity and expression level of IDP transcripts, and how this links to levels of disease pathogenesis. This, along with ongoing eDNA monitoring by collaborators in the UK and at Yellowstone, will help refine serological approaches to track the demographics of the most virulent parasite genotypes. The work will contribute towards the development of important disease control strategies for fisheries managers and government agencies in pre-empting future disease outbreaks.
We are aiming to work closely with CASE partner Pacific Seafoods who have experienced severe seasonal PKD outbreaks since 2017. They have been working with the US. Geological Survey (USGS), US Department of Agriculture (USDA), the Washington State Department of Fish & Wildlife (WDFW) and the Colville Confederated Tribe (CCT) to understand the parasite that causes PKD and how it affects farmed fish. Designated sampling sites will be net pen sites downstream of the Grand Coulee Dam and upstream of the Chief Joseph Dam on the Columbia River, Washington. Wild fish samplings will be undertaken above and below the Grand Coulee Dam. The USGS has been studying PKD outbreaks in the Yellowstone River (Montana) for several years, developing PCR diagnostics, eDNA monitoring and genotyping of fish and parasite populations facilitated by mark-recapture studies. Existing bioarchives covering both rivers along with hydrological / temperature profiles, and eDNA datasets will provide the opportunity to explore links between ecological and environmental parameters during the PKD season with strain diversity and parasite virulence. Yellowstone sampling sites will be at Livingston and Emigrant, Montana.
We have well established links with Trafalgar Fisheries, Test Valley Trout Ltd, and the British Trout Association who will facilitate fish sampling from UK rivers endemic for PKD. We also have a well-established link with Vertebrate Antibodies Ltd (VAL) at UoA in generating antibodies and development / refinement of TbMEG-1 serological tools. VAL will provide in-kind support in refining our approach for testing serum samples from whitefish populations in Montana as well as trout populations within UK and USA rivers.
This project represents world-leading applied research that is aligned with the NERC science remit and QUADRAT themes in developing enhanced tools and management practices for safeguarding natural and managed freshwater aquatic environments in the face of climate change. The project has very strong potential to help safeguard UK fish biodiversity against PKD by addressing adaptive parasite virulence factor diversity in wild populations currently impacted by climate change-driven ecological and environmental processes. Taken together, this work is closely aligned with NERC’s research priority to address applied challenges of managing and building resilience to environmental change in natural and managed resources.
Project Aims:
- Identify IDP sequence diversity correlating with parasite strains varying in virulence within and between fish host species.
- Determine relationships between IDPs linked to distinct parasite strains and virulence levels with host / parasite densities, parasite kidney load, and fish immunological status.
- Determine how relationships between parasite strain / virulence levels and IDP expression profiles are affected by ecological and environmental factors during PKD seasons.
These will be achieved by:
- Shortlisting of secretory IDPs from existing T. bryosalmonae genome using our antigen selection pipeline. Further selection based on fish specificity by q-PCR using existing fish and invertebrate host samples.
- Samples from trout populations in UK rivers and from the Columbia River and from whitefish populations in Yellowstone will be available. Fish will be selected for sequencing based on dominant parasite strain identity (from microsatellite markers), parasite load and pathogenicity scored histologically and from kidney swelling index data. RNA and DNA will be extracted from infected fish kidney for transcriptional profiling, parasite load and verification of parasite genotype. Serum samples will be used to establish fish immunological status by indirect ELISA.
- Determining IDP sequence diversity and expression profiles will be achieved by targeted RNA-Seq. 80 RNA samples will be used to capture 50-100 IDPs. Captured pair-end reads will be assembled and analysed in Trinity followed by DGE analysis. IDPs strongly correlating to specific parasite strains and/or virulence levels will be analysed by q-PCR.
- Parasite and host densities from eDNA monitoring, hydrological and water temperature data will be available. Multivariate statistics will be used to identify correlations between transcript diversity / expression level, strain diversity, virulence level, and fish immunological status. How biotic factors are influenced by changing ecological and environmental parameters prior to and during the PKD season will also be explored.
Essential & desirable candidate skills
Essential: Applicants should hold at least a 2:1 honours degree in biological sciences, such as environmental sciences, ecology, and molecular biology. Applicants with a first-class degree and/or a master’s qualification are particularly encouraged to apply.
Desirable: Experience in the following is desirable: Molecular biological techniques; Statistical analysis using R or equivalent; Field work studies; Bioinformatics, including Linux OS and RNA-Seq analysis pipelines
Supervisors
Jason HollandPrimary Supervisor: | Profile: Jason Holland Email: j.holland@abdn.ac.uk Institution: University of Aberdeen Department/School: School of Biological Sciences |
Caroline MehargSecondary Supervisor: | Profile: Caroline Meharg Email: caroline.meharg@qub.ac.uk Institution: Queen's University, Belfast Department/School: School of Biological Sciences |
Marius WenzelAdditional Supervisor: | Profile: Marius Wenzel Email: marius.wenzel@abdn.ac.uk Institution: University of Aberdeen Department/School: School of Biological Sciences |
References
Faber, M., Shaw, S., Yoon, S., De Paiva Alves, E., Wang, B., Qi, Z., Okamura, B., Hartikainen, H., Secombes, C. J., Holland, J.W. (2021) Comparative transcriptomics and host-specific parasite gene expression profiles inform on drivers of proliferative kidney disease. Scientific Reports, 11, 2149.
Bartošová-Sojková, P., Kyslík, J., Alama-Bermejo, G., Hartigan, A., Atkinson, S. D., Bartholomew, J. L., Picard-Sánchez, A., Palenzuela, O., Faber, M. N., Holland, J. W., Holzer, A. S. (2021) Evolutionary Analysis of Cystatins of Early-Emerging Metazoans Reveals a Novel Subtype in Parasitic Cnidarians. Biology, 10, no.2, 110.
Abos, B., Estensoro, I., Perdiguero, P., Faber, M., Yehfang, H., Diaz Rosales, PD., Granja, AG., Secombes, C., Holland, JW. Tafalla, C. (2018). Dysregulation of B cell activity during Proliferative Kidney Disease (PKD) in rainbow trout. Frontiers in Immunology 9, Article 1203
Expected Training Provision
This project provides extensive molecular biology training and opportunities to engage in advanced bioinformatics. A proposed 15-week lab and fieldwork trip to the Columbia River (Washington) and the Yellowstone National Park (Montana) in the USA will be included. The student will be registered at the University of Aberdeen and will be able to take full advantage of all multi-disciplinary training opportunities available. Specifically, the student will be trained in bioinformatic techniques and multivariate statistics, including the use of the command line in R. Such skills will be required in utilizing in silico antigen pipelines to shortlist IDPs from transcriptome / genome assemblies, to implement targeted (capture) RNA-Seq, and in determining the statistical relationships between multiple datasets. Training will be provided by both project supervisors and through provision of workshops / courses and other training opportunities via the Centre for Genome Enabled Biology and Medicine at Aberdeen (CGEBM) and The Genomics Core Technology Unit (GCTU) at Queens University Belfast.
Full training opportunities will be provided in laboratory molecular biology techniques, including nucleic acid purification from tissue samples and primer design for PCR, and q-PCR analysis, histology / immunohistochemistry for antigen detection in fixed tissues, and in the use of indirect ELISAs to monitor antigen-specific antibody responses in fish serum samples. The student will work alongside Vertebrate Antibodies Ltd to get first-hand insights into the production and testing of monoclonal antibodies and techniques used in developing recombinant protein and protein epitope based indirect ELISAs.
The student will undergo a Home Office SCOTPIL training course to gain a personal licence to undertake procedures covered by the Animals (Scientific Procedures) Act (fish tagging and collection of fish blood and other tissue samples). This will be complemented with the opportunity to attend an international course on fish immunology held annually at the University of Wageningen, Netherlands.
As part of the proposed 15-week visit to our US project partners, the student will work alongside Pacific Seafoods, USGS and collaborating agency personnel in participating in lab and fieldwork studies gaining valuable skills in sample and data collection, molecular and histological diagnostics, eDNA monitoring by q-PCR. Other training opportunities will be available in field techniques, including electrofishing, fish handling and sampling techniques, and set up of mark-recapture experiments to monitor individual fish over time. Laboratory training will include sample preparation and strain / genotyping of parasites infecting sampled fish. The student will also benefit from training and sampling opportunities with our UK partners. Other training opportunities will be available at the University of Aberdeen and via comprehensive practical and classroom-based QUADRAT training opportunities in developing core and generic skills (eg. communication, written, and presentational skills) in disseminating research findings at project meetings and National and International conferences.
Impact
Myxozoan parasites belong to a highly divergent yet morphological simplified parasitic group within the Phylum Cnidaria and, thus, represent some of the most primitive of metazoan parasites. Their uniqueness is exemplified by the discovery and functional characterization of the secretory IDP, TbMEG-1, now known to represent the first non-helminth microexon gene. Its ability to act as self translocating effector protein of fish lymphoid cells is a paradigm shift in our understanding of such effector proteins given that no other metazoan group expresses them. TbMEG-1 has also exhibited partial protection as a DNA- and recombinant protein-based vaccine for PKD. This project provides the opportunity to explore the role of this antigen in immune evasion and virulence in wild fish populations of economic and conservation importance.
Our proposed work will provide a baseline for the characterization of additional fish specific secretory IDPs. This will contribute to the understanding of myxozoan virulence and provide additional targets for therapeutic studies in other related disease agents. The development of the first non-invasive means to serologically monitor parasite exposure and immunological status of trout species via an indirect ELISA lends itself to commercial development (dipstick serologic test for in-the-field assessment). Along with Vertebrate Antibodies Ltd, we have shown that indirect ELISA protocols can be refined using protein epitopes. If, in this project, we can link transcript repertoires and / or alternatively spliced isoforms with high virulence levels, it will be possible to develop an epitope based indirect ELISA specific for the detection of the most virulent parasite strains. This would provide a powerful means to monitor and mitigate disease spread.
Proposed Supervision
The supervisory team encompasses a wealth of experience in functional genomics, bioinformatic experience, and host-pathogen interactions with specific laboratory-based expertise in the study of PKD. In utilizing existing bioinformatic pipelines and transcriptome / genome assemblies, in-depth knowledge of command line-based approaches will ensure the student receives full guidance and training in antigen selection, targeted RNA-Seq and statistical approaches. Dr Jason Holland will be the designated project lead with specific expertise in the study of PKD that incorporates an existing network of collaborative links with academia, government agencies and industry stakeholders within the UK, Europe, and USA. Dr Marius Wenzel has expertise in functional (epi) genomics, microbiomics, bioinformatics, and host-parasite interactions and Dr Caroline Meharg has expertise in functional genomics, bioinformatics, environmental microbiology, and host-microbe interactions. US CASE and non-CASE partners will oversee lab and fieldwork studies during the 15-week student visit to the USA. The Aberdeen supervisors and non-CASE partner will jointly oversee the indirect ELISA studies.
The supervisory team will agree a timetabled programme of research (computer-, lab-based and written work) activities and project milestones with the student and a development programme that will include all training and skills required to fulfill project goals. Both will be reviewed as the project progresses. Other professional development opportunities will be discussed, including appropriate workshops, conferences, and outreach events. Dr Holland will be available for day-to-day project advice and guidance and will ensure smooth planning and execution of USA-based fieldwork studies. Aberdeen supervisors will meet with the student bi-weekly complemented with monthly full supervisory meetings. More formal meetings to discuss progress against milestones will be carried out 6-monthly. Regular contact with all supervisors will be done in person, phone contact and via email with mutual arrangements made with the supervisors regarding the timing and nature of contact.
Proposed Timetable
M1-M12: The project will begin with in-silico selection of secretory IDP candidates. Further selection will be limited to those deemed to be fish-specific at the transcriptional level by
q-PCR analysis. To complement existing tissue sample archives held by US project partner, a 15-week lab and fieldwork study visit to Washington and Montana will be undertaken during the Spring-Summer of Year-1.
M13-M24: Project partner, Vertebrate Antibodies Ltd will undertake production of a monoclonal antibody to mountain whitefish IgM, enabling the monitoring of antigen-specific antibody responses by indirect ELISA in Year-3. Existing mabs to salmonid IgM will be used to monitor antibody responses in trout species. Sample processing and nucleic acid purification will be undertaken and, once quality checked, used for parasite genotyping, and targeted RNA-Seq library preparation and sequencing (to be carried out by CGEBM). Transcript assembly will be undertaken using our in-house parasite genome assembly. Archived formalin fixed tissue samples will be processed for histological and immunohistochemical studies using existing TbMEG-1 antibodies.
M25-M36: Analysis of parasite genotyping and targeted RNA-Seq datasets and begin multivariate statistical analysis of all assimilated data. Undertake indirect ELISA studies that will be benchmarked with the existing TbMEG-1 assay based on full length recombinant TbMEG-1 with further development using specific protein epitopes from TbMEG-1 / IDP RNA-Seq data associated with high virulence levels.
M25-M42: Drafting of manuscripts
M36-M42: Final data analysis and thesis drafting / submission.
QUADRAT Themes
- biodiversity
- environmental-management
Partners
A CASE partnership is under discussion with Pacific Seafood Group. An update will be provided in due course.
Non-CASE partnerships have been confirmed with the following organisations:
Vertebrate Antibodies Ltd
Test Valley Trout Ltd
British Trout Association
U.S Geological Survey – to be confirmed
