Biodiversity, phylogeny, and ecology of large naked ramose/reticulose amoebae: barely studied but diverse and common components of many ecosystems

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

Abstract

Recent advances in molecular biology techniques and analyses have greatly advanced the evolutionary and ecological study of microbial eukaryotes (protists). Because their appearance often does not reflect their evolutionary relationships, protist groups can generally only be properly understood when we have both morphological and genetic data and can use phylogenetic analyses to reconstruct their evolutionary relationships. Many protist groups have recently been elucidated in this way. In addition, related molecular techniques have been used to show that the genetic diversity of protists in most environments is massive - many more organisms await discovery than are known morphologically. However, one group of protists that has largely eluded both culture-based and molecular studies is that of large, non-shelled amoebae with long, often branching extensions to their cells (ramose pseudopodia), some of which fuse to form cytoplasmic 'networks' (reticulopodia) of varying sizes. We call them NRRA - naked ramose/reticulose amoebae. NRRA may be almost stationary, attached to surfaces or extending their pseudopodia between soil or sediment particles, or they can be active predators, moving across surfaces or through particulate material in search of prey. There are not many cultured NRRA because they are difficult to grow, but all known strains feed voraciously on a wide range of food sources - other protozoa, unicellular and multicellular algae, many types of fungi including those known to cause diseases of crops or other plants, and even small animals like nematode worms and rotifers. It is possible that some attack even larger organisms. Therefore, NRRA are likely to be important agents of biological control, influencing other microbial activity including disease-causing organisms and those involved in decomposition and nutrient cycling. NRRA remain very poorly known because their delicate cell shape means they are often destroyed by sampling techniques. They take a long time to appear in environmental samples in the laboratory and often need particular (but usually unknown!) conditions to grow. Furthermore, they are relatively rarely detected by molecular studies because their versions of the genes used for such studies are often so different from many other organisms that the standard molecular probes fail to detect them. The aim of our project is to find more NRRA in a wide range of habitats and describe their morphologies and lifecycles, and to measure how big their appetites are - both in terms of the diversity of their diet and how much of it they eat (including fungi and other organisms which are known to cause diseases of higher plants). We will sequence genes from NRRA cells in order to understand their evolutionary relationships and histories. We will measure how diverse they are (how many 'species', although this is often an unclear concept in many protists) under many different environmental conditions. Taking this line of enquiry further, we will collaborate with three research programmes that run long-term experiments on 1) forest soils, 2) agricultural vs. unploughed and uncultivated soils, and 3) freshwater lakes. These experiments have well established sampling strategies and other data collected from the sample sites, which will provide a very informative context for our new data on NRRA. By intensively sampling sites contributing to all three experiments we will investigate whether changes in NRRA diversity and abundance correlate with changes in other microbial (and small animal and plant) groups. If they do we will suggest possible reasons for the correlations, which can be tested in future studies. We will also work out the environmental conditions (temperature, pH, amount of dead organic material, etc.) under which NRRA are most diverse and abundant. These results combined will help scientists and environmental agencies to assess the 'health' and carbon budgets of soil and freshwater environments.

Publications

10 25 50
 
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. We have discovered and described six new genera of large network-forming amoebae (NFA), and shown them to be dominant members of soil habitats. NFA are involved in a wide range of feeding levels, able to rapidly consume a diversity of prey from bacteria up to small animals (nematodes, rotifers, etc.).
2. Another discovery was the abundant and diverse marine amoeba Reticulamoeba, which we sequenced and placed phylogenetically, and proved that although a very frequent member of marine sediment habitats it is never or very rarely detected by large-scale high throughput sequencing surveys that are undertaken on a global scale, because it's genes are too evolutionarily divergent to be detected using normal methods.
3. We are currently using over a billion sequences from a soil gene expression experiment to understand the contribution of NRA to soil processes in the context of other eukaryotes and bacteria.
4. We show that NFA are important members of many ecosystems but have been overlooked or missed by previous studies because they are difficult to grow in culture and often difficult to analyse or detect genetically. This we emphasise that a multidisciplinary and integrated approach is needed to more fully understand microbial biodiversity patterns and ecology.
5. We revealed a large diversity of vampire amoebae (vampyrellids) including many new lineages and new species in marine habitats, where they were previously almost unknown.
6. We discovered and are in the process of analyzing several novel NFA lineages that represent apparently highly divergent eukaryotic lineages, and require complex analyses to understand their evolutionary links.
Exploitation Route Our results provide a range of insights into eukaryote evolution, which will inform and be intergrated into developing evolutionary models, being addressed by researchers worldwide. We also show that network-forming amoebae are more important and diverse elements particularly in soil systems than previously recognised, and are working with Rothamsted Research to more elucidate more clearly their roles in soils. This includes measuring their previously hypothesised roles in disease suppressivity of soils. The results will be potentially applicable for soil remediation work - i.e. supplementing soils to encourage the activity of NRRA.
Sectors Agriculture, Food and Drink,Education,Environment

 
Description Although still quite early days, the work on large network-forming amoebae is being integrated into understanding of soil microbial diversity and function, particularly via collaboration with Rothamsted Research, and marine systems via BioMarKs and other marine-based collaborations. Also eukaryotic evolution studies, particularly relating to the many variosean amoebae (Amoebozoa) that we detected and described. Impacts will develop over the next few years.
First Year Of Impact 2012
Sector Agriculture, Food and Drink,Education,Environment
Impact Types Cultural

 
Description BBSRC CCC Scheme
Amount £12,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start  
 
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
 
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 NHM MCf 
Description The Molecular Collections facility (MCf) at the Natural History Museum is a large, curated cryopreservation facility that curates samples (frozen tissues, organisms, nucleic acids, etc.) for use by other researchers and collaborators worldwide. When currently ongoing work is completed, the DNA samples produced during these grants will be deposited in the NHM MCf with associated metadata for such use. The two projects reported here have generated at least 2000 samples of DNA extracted from many environmental matrix types, host organisms (for parasites), and microcosm/other experimental systems. 
Type Of Material Database/Collection of data 
Year Produced 2011 
Provided To Others? Yes  
Impact Academic publications (e.g. Simek K, Kasalický V, Jezbera J, Hornák K, Nedoma J, Hahn MW, Bass D, Jost S, Boenigk J. (2013) Differential freshwater flagellate community response to bacterial food quality with a focus on Limnohabitans bacteria. ISME Journal) and developing collaborations. 
 
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 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 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 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 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 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 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