Ecological and morphological characterisation, and phylogeny of Endomyxa: poorly known but widespread and diverse protozoan parasites and omnivores

Lead Research Organisation: Natural History Museum
Department Name: Life Sciences

Abstract

Endomyxa are a very poorly known but large and diverse group of organisms in the protozoan phylum Cercozoa. Endomyxa includes the commercially important plasmodiophorid plant parasites, and haplosporidia and relatives, which include parasites of a wide range of invertebrates, most famously MSX disease of oysters. The evolutionary relationships of both of these groups were for a long time unknown (plasmodiophorids were considered fungi for a long time), but good ribosomal DNA trees show that they are related to each other (a relationship reinforced by morphological synapomorphies) and several lines of molecular evidence show that they belong within the eukaryotic supergroup Rhizaria, specifically as a subphylum within Cercozoa, but separately from the so-called 'core' Cercozoa. More recently, free-living relatives of these parasites have been found through culturing/cell isolation and rDNA sequencing. These include the large testate marine filose amoeba Gromia, and large reticulose, naked amoebae: the bacterivorous Filoreta and the predatory Arachnula and Platyreta. Such organisms represent very distinct and poorly studied protozoan morphotypes whose ecological roles are almost completely unknown. Other research has revealed more endomyxan parasites, for example the spot prawn parasite, and Paradinium, a parasite of copepods. Culture-independent environmental rDNA libraries show that there are a large diversity of other endomyxan clades and lineages which remain uncharacterised - the only information we have for them is the provenance of the samples in which the sequences were detected. The environmental libraries also show that there is very strong ecological structuring - many endomyxan clades have so far only been found in quite specific habitats, for example deep-sea samples, anaerobic marine, anaerobic freshwater, or phylloplane communities. These patterns suggest high levels of ecological specialization, perhaps involving interactions with other organisms. This project aims to identify as many of these novel lineages and clades as possible, whether they are free-living, symbiotic, or parasitic, using a combination of intensive selective culturing using a diversity of food sources and culture conditions informed by the results of the environmental libraries, and fluorescent in situ hybridization (FISH) using fluorescent probes that will specifically detect chosen uncharacterized lineages. FISH will also show the relative abundance of endomyxan lineages in different habitats, and whether they are associated with, or are found inside, other eukaryote cells, and if so, which organisms they co-occur with (and are therefore perhaps parasites of). Multiple and diverse SSU rDNA environmental libraries will be constructed, using a set of overlapping PCR primers covering all Endomyxa, to show much more clearly and realistically than previously possible the true diversity of Endomyxa, and their ecological nature by analyzing the sequence data in the context of other biotic and abiotic variables co-measured at each sampling site. The libraries will be made using both rDNA and crDNA reverse transcribed from rRNA: the latter is generally taken as a surrogate for the level of activity of the cells (more ribosomes will be present in more active cells), rather than just presence/absence and biomass as indicated by rDNA. This approach will provide more informative about the ecological characteristics than rDNA libraries alone. The environmental sequences will be used to improve phylogenetic reconstructions of Endomyxa and their relationships to the rest of Rhizaria both by increasing taxon sampling of this part of the tree, and by providing sequence data that can be used to obtain LSU rDNA sequences for representative genotypes directly from environmental DNA samples. Where cultures are available, other genes can be targeted by PCR, to be combined with existing data in multigene phylogenies.

Publications

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Adl S (2019) Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes in Journal of Eukaryotic Microbiology

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Adl SM (2012) The revised classification of eukaryotes. in The Journal of eukaryotic microbiology

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Bass D (2018) Clarifying the Relationships between Microsporidia and Cryptomycota. in The Journal of eukaryotic microbiology

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Bass D (2019) Ascetosporea. in Current biology : CB

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Bass D (2019) The Pathobiome in Animal and Plant Diseases. in Trends in ecology & evolution

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Bass D (2021) Parasites, pathogens, and other symbionts of copepods. in Trends in parasitology

 
Title Black Box Safari 
Description An art installation in Oxfordshire where members of the public were offered a self-guided tour (safari) where black boxes were distributed around the site. Each box had a magnifying glass attached to the top of the box, which invited insepction. On looking through the glass, viewers see a loop video showing eukaryotic microbes filmed from samples taken from the same site. 
Type Of Art Artistic/Creative Exhibition 
Year Produced 2011 
Impact Many discussions on microbial biology, eukaryotic evolution, the interaction of art an science, and the roles of scientists in society. An extremely and often unexpectedly productive and rewarding set of interactions. 
 
Description 1. Demonstration and analysis of large diversity of Endomyxa in many globally distributed locations and the majority of habitat types.
2. Reconstruction of the endomyxan evolutionary tree, showing how different body forms, lifestyles (e.g. parasitism, predation, large body size) have evolved and from which ancestors.
3) Description of new endomyxan taxa (species and above), including both parasites and free-living organisms.
4) Discovery and characterisation of a major new group of parasites - the mikrocytids - which cause serious diseases of crabs and bivalve molluscs that impact the fishing industry, and demonstration that these parasites are diverse and sometimes abundant in a wide range of hosts in both marine and non-marine habitats.
5) Discovery of previously hidden diversity of plasmodiophorid plant parasites and their relatives that parasitise many marine algae, seaweeds, and seagrasses. Our analyses showed very wide variation in host taxa for these parasites, suggesting their general occurrence and importance.
6) Unveiling of large diversity of vampire amoebae (vampyrellids) including many new lineages and new species in marine habitats, where they were previously almost unknown.
7) Development of environmental DNA methods for detecting and discovering microbial diversity and understanding its ecology without having to sample or culture individual cells, which is time consuming, expensive, and scientifically biased.
Exploitation Route Our findings on parasites are being integrated into work on shellfish of food security interest, both in terms of understanding diseases of these food organisms and how fisheries may be sited to avoid such diseases, and also methods of detecting parasites and pathogens in environmental samples. The latter is potentially very powerful as it makes sampling less biased, higher throughput, and therefore more efficient and less expensive, but has a requirement for specificity that is informed by our findings about high levels of diversity of these parasites in many habitats.
Sectors Aerospace, Defence and Marine,Agriculture, Food and Drink,Education,Environment,Government, Democracy and Justice

 
Description Discoveries of novel parasitic taxa and diversity are currently being used in a food security context - i.e. in understanding the role of these novel parasites and diversity of pathogens is a) directly affecting shellfish health and survival, and b) methods of monitoring disease risk and occurence and policies related to those activities, including many developments of eDNA methods.
First Year Of Impact 2012
Sector Aerospace, Defence and Marine,Agriculture, Food and Drink,Education,Environment,Government, Democracy and Justice,Culture, Heritage, Museums and Collections
Impact Types Cultural,Economic,Policy & public services

 
Description Parasite diversity and distribution
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Systematics in parasitology
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
 
Description BBSRC CCC Scheme
Amount £12,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start  
 
Description BBSRC Newton Fund GRP
Amount £1,900,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2016 
End 02/2019
 
Description BBSRC SARISA 2014
Amount £30,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description Defra research and development project
Amount £546,480 (GBP)
Organisation Department For Environment, Food And Rural Affairs (DEFRA) 
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2020
 
Description H2020-SFS-2015-2
Amount € 449,000 (EUR)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 04/2016 
End 03/2020
 
Description NERC Freshwater Highlight topic
Amount £552,000 (GBP)
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 04/2016 
End 04/2020
 
Description Research Project
Amount £750,000 (GBP)
Organisation Department For Environment, Food And Rural Affairs (DEFRA) 
Sector Public
Country United Kingdom
Start 04/2015 
End 03/2018
 
Description SynTax (Phytomyxids)
Amount £19,845 (GBP)
Organisation Linnean Society of London 
Sector Charity/Non Profit
Country United Kingdom
Start  
End 12/2014
 
Description SynTax (parasites)
Amount £29,000 (GBP)
Organisation Linnean Society of London 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2013 
End 02/2014
 
Title Parasite environmental detection and non-invasive infection status assessment by eDNA 
Description The use of eDNA methods (nucleic acid extraction from environmental or organismal matrix following by sequencing either via general or specific PCR primers or by metagenome/transcriptome sequencing) has been shown to be a powerful method for measuring microbial (including parasite) diversity and distribution in situ. We have refined these sampling and processing methods for eukaryotic aquatic parasites, reported in several papers, and others in prep. Additionally, we have developed and are refining non-invasive eDNA-based methods for detecting and monitoring disease status of shellfish and finfish for use in aqaculture and wild capture fisheries research and monitoring. 
Type Of Material Biological samples 
Year Produced 2012 
Provided To Others? Yes  
Impact Academic publications, (pending) adoption of sampling and processing methods into workflow of relevant practitioners (e.g; Cefas, Environment Agency, JNCC). See details of workshop delivery to JNCC in other section of this form. Similar delivery to Cefas. 
 
Title Cefas collections 
Description Cefas maintains collections of parasite(-related) material. Much of the parasite work as part of the Endomyxa NERC award was done in collaboration with Cefas, resulting in their adding histology slides, EM preps, and fixed specimens for further analysis to their collections. These correspond to crustean, mollusc, and annelid hosts, and a range of microbial eukaryotic parasites, mostly ascetosporeans (Endomyxa). 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact The edible crab materials for Paramikrocytos canceri, a newly described mikrocytid crab pathogen have been used in a Current Biology (2014) paper on this organism and other mikrocytids, and have been made available to other researchers collaborating with us on mikrocytid genomics. The collections will also be used by national reference laboratories across Europe (EURLs), within which Cefas is the crustacean representative. 
 
Title Protist culture collections 
Description Protist strains isolated and named were deposited in several international culture collections - CCAP, ATCC, NIES 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? Yes  
Impact Protist strains provided directly to other researchers for taxonomic, ecological, and cell biology studies and publications. Making these available also via publically-accessible culture collections. 
 
Description BioMarKs 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Biological Station, Roscoff
Country France 
Sector Academic/University 
PI Contribution Contribution to sampling and processing of samples and data. Specific scientific input regarding Cercozoa (including Endomyxa) diversity analyses, phylogenetics, and interpretation.
Collaborator Contribution Covering the cost for and carrying out 1.5 plates of 454 sequencing to investigate endomxyan diversity in marine sample. Data generated, analyses ongoing.
Impact Publications listed in relevant section. Contribution to seminars and scientific meetings connected with this collaboration. Other aspects of work in progress.
Start Year 2009
 
Description Cefas 
Organisation Centre For Environment, Fisheries And Aquaculture Science
Country United Kingdom 
Sector Public 
PI Contribution Expertise in molecular protistology, eDNA methods, and phylogenetics, contributing to a balanced collaboration.
Collaborator Contribution Expertise in pathology, parasitology, and associated microscopy, contributing to a balanced collaboration.
Impact Several publications on protistan parasites - see publications section. Also student co-supervision, including a NERC DTP (GW4+) studentship with Exeter and The Natural History Museum, starting September 2014. The collaboration is multi-disciplinary, involving next generation sequencing, eDNA, phylogenetics, parasitology, pathology, histology, electronc microscopy, and policy engagement.
Start Year 2011
 
Description Collaboration with Oslo University 
Organisation University of Oslo
Country Norway 
Sector Academic/University 
PI Contribution Heavy contribution to funding proposal to Research Council of Norway on X-cell (alveolates) parasites of fish, and subsequent meetings, collaboration, analyses, paper writing, etc.
Collaborator Contribution Managing funded grant, collaboration, next generation sequencing (amplicon and metagenomics), analyses, paper writing, etc.
Impact Large sequence datasets generated from fish and invertebrate hosts, environmental samples, etc. - both amplicon and metagenomics libraries. Publications in preparation.
Start Year 2011
 
Description NERC PhD Studentship, Warwick 
Organisation University of Warwick
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution David Bass devised as is co-supervisor on a NERC-funded CASE PhD studentship held at Warwick School of Life Science, with NHM as CASE partner. The project is on plasmodophorid plant parasites. DB's contributed is training and supervision relating to this particular group of protists (belonging to Endomyxa), molecular phylogenetics, bioinformatics, microbial ecology, and eDNA methods. The NHM also co-funds the studentship (stipend and scientific costs).
Collaborator Contribution Warwick hosts the student, provides expertise on soil ecology and plant-soil-microbe interactions, and statistics.
Impact Training of PhD student to delivery of PhD thesis (due in 2015), papers in preparation.
Start Year 2011
 
Description Rothamsted Research 
Organisation Rothamsted Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Data analysis for soil metatranscriptome project and sequencing costs for soil disease suppressivity experiments.
Collaborator Contribution Large scale soil transcriptome data analysis (in partnership with TGAC Norwich), and provision of samples for this project and disease suppressivity project. Several papers in preparation.
Impact Collaboration and publications in preparation
Start Year 2011
 
Description August 2019: Past President's Plenary address at ISoP, Rome 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary presentation at the annual meeting of the International Society of Protistologists, in role of Past President
Year(s) Of Engagement Activity 2019
 
Description Co-organiser and plenary presenter, ARCH-UK Microbiome Workshop, Aberdeen 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Workshop as part of ARCH-UK NERC/BBSRC Aquaculture initiative. Presentation on microbiomes followed by discussions, session chairing, and workshop leading.
Year(s) Of Engagement Activity 2018
 
Description February 2019: Invited seminar, Oslo University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Research presentation with collaborators and wider audience in Oslo University Biosciences department
Year(s) Of Engagement Activity 2019
 
Description Filming for BBC 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview for BBC4 programme (Nature's Weirdest) on use of next generation sequencing methods for biological discovery and characterisation, based on methods (metagenomic sequencing) used in both NERC grants.

Programme to air in January 2015 so no further impact yet.
Year(s) Of Engagement Activity 2014
 
Description Invited plenary speaker at Czech Society for Parasitology meeting, Czech Republic 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary talk at parasitology conference followed by discussions.
Year(s) Of Engagement Activity 2018
 
Description Invited plenary speaker at Society for Experimental Biology meeting, Azores 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited presentation at conference focussing on effects of high CO2 levels on microbes and influencing animal disease in the oceans.
Year(s) Of Engagement Activity 2018
 
Description Invited seminar, Oslo University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Presentation to collaborators and other faculty members in the biosciences department of Oslo University. Approx 40 attendees.
Year(s) Of Engagement Activity 2019
 
Description March 2019: Symposium chair: eDNA, ecology and environment, World Aquaculture Society meeting, New Orleans 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talks and workshops in eDNA and parasites/diseases of aquatic animals given at World Aquaculture Society conference
Year(s) Of Engagement Activity 2019
 
Description Postgraduate teaching 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Teaching postgraduates about microbial biodiversity and evolution, and next generation sequencing technologies and applications. Also parasite-host interactions and parasite ecology.

Discussion, MSc, MRes, and PhD project students, publications, requests for advice and assistance.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014
 
Description School talk - Kingsbridge Community College 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk to school students (GCSE to A level) on symbiosis and parasitism. Subsequent delivery of annotated slides and follow-up contact/advice.
Year(s) Of Engagement Activity 2015
 
Description Schools talks 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk to GSCE and A level students followed by questions and discussion, and more generally on career progression and choices in science.

Post-talk discussion, work placement students
Year(s) Of Engagement Activity 2012,2013,2014
 
Description Science Uncovered 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Science Uncovered event at The Natural History Museum (NHM), attended by c. 10,000 people - part of European Researchers' Night (EU funded). Showcasing the science and research at NHM. I contributed to two stalls - one of microeukaryotic diversity, the other on the SARISA (BBSRC) Roots of Decline project.
Year(s) Of Engagement Activity 2016
URL http://www.nhm.ac.uk/visit/exhibitions/science-uncovered-2016.html
 
Description Science Uncovered, NHM 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Science Uncovered at The Natural History Museum, and other public engagement events at NHM. Demonstration and discussion of work relating to these grants and more broadly.

Lab visits, discussion, student placements, requests for advice.
Year(s) Of Engagement Activity 2011,2012,2013
 
Description Symposium chair: eDNA, ecology and environment, World Aquaculture Society meeting New Orleans 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 40 people in symposium (eDNA, ecology and environment, World Aquaculture Society meeting, New Orleans). 5 talks followed by discussion and information sharing.
Year(s) Of Engagement Activity 2019
 
Description Talk to Fleet Study Group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Talk given on microbiakl eukaryote diversity and role of parasites to the Fleet Study Group, Weymouth, in relation to their conservation and management work on Chesil Beach and the Fleet lagoon.

Requests for advice re. eDNA methods, questions about the science presented, interest in future collaboration/consultancy.
Year(s) Of Engagement Activity 2013
 
Description VIVALDI H2020 bivalve health project conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary presentation and session coordination for EU-funded bivalve health and disease mitigation project.
Year(s) Of Engagement Activity 2019
URL https://www.vivaldi-project.eu/
 
Description Workshop for JNCC 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited workshop on eDNA methods to staff/practitioners at JNCC. Devised and presented by David Bass and Hanna Hartikainen (worked on Endomyxa NERC grant). Seminars and workshop exercises. Presentation of materials to JNCC following the 1-day event.

Greatly increased understanding of these molecular methods by JNCC staff and discussion about how they could be applied. Ongoing integration and development of eDNA methods into JNCC work.
Year(s) Of Engagement Activity 2014
 
Description eDNA in Parasitology workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact workshop on the use of Edna (environmental DNA) methods in parasitology and policy development.
Year(s) Of Engagement Activity 2015