Methods summary: Applying CRISPR to detect eDNA

Article by Molly-Ann Williams and Anne Parle-McDermott both from Dublin City University

We were challenged to design and build a simple and rapid species monitoring system. Why do we need such a system?  Biodiversity loss is at an all-time high and such a system would help to support the management and conservation of fish species within aquatic environments by acquiring knowledge of species distribution that traditionally is gained through visual detection and counting. These methods are expensive, time consuming and can lead to harm of the species of interest.    We decided that environmental DNA (eDNA) was the way to go but we had to solve the ‘PCR problem’ i.e., avoid having to do cyclical high temperatures as that would see us ending up with a costly, once-off device that would likely not be applied outside our lab.  This got us brainstorming and led us to a novel isothermal detection method, combining Recombinase Polymerase Amplification with CRISPR-Cas detection, which simplifies the adaptation of nucleic acid detection on to a biosensor device.

This innovative methodology utilises the collateral cleavage activity of Cas12a, a ribonuclease guided by a highly specific single CRISPR RNA, to detect specific species from eDNA. We proved it could work for eDNA by applying the technology to the detection of Salmo salar from eDNA samples collected in Irish rivers, where presence or absence had been previously confirmed using conventional field sampling. The beauty of this advance is that it can be applied to any species in the environment.  Not only does this assay solve the ‘PCR problem’, it is also is a better approach for distinguishing very closely related species.  We look forward to others in the field adapting it to their own favourite species of interest.  

Citation: Williams, M‐A, O’Grady, J, Ball, B, et al. The application of CRISPR‐Cas for single species identification from environmental DNA. Mol Ecol Resour. 2019; 19: 1106– 1114. https://doi.org/10.1111/1755-0998.13045

Interview with the author: Sociality, hyenas and DNA methylation

Adding of methyl groups to a DNA molecule or methylation has the interesting ability to alter the activity of a DNA segment without changing the sequence.  In this behind the scenes look, Zachary Laubach and colleagues test if this valuable biomarker is impacted by differences in hyena social status or other ecological factors early in life. What’s particularly impressive is that they garnered insights into methylation from a wild population. They find some surprising results, such as that high ranking mums can confer higher levels of methylation to their cubs that disappears when they get older. Why? Find out below and read the full article here.

Photo credit: Zach Laubach

What led to your interest in this topic / what was the motivation for this study? 

Across a broad taxonomic spectrum, social experiences, particularly those early in life, seem to have a profound impact on organisms’ development. The idea that during sensitive periods of development, social experiences and early life environment can have lasting impacts on the later life phenotype and health is known as the Developmental Origins of Health and Disease (DOHaD) hypothesis, and was formalized in the 1980s by epidemiologists, namely David Barker and his research on cardiovascular disease. Among social mammals, including humans and non-human primates, an individual’s social rank affects their behavior, physiology, and related health outcomes. For example, in humans, low socioeconomic status is widely recognized as a risk factor for cardiovascular complications and other chronic diseases. In non-human primates, low social rank is risk factor for elevated chronic stress and immune dysregulation. So, although we observe that social status affects biology, we still know little about how this all works. To better understand a potential mechanism for how early life environment affects biology, we investigated possible early environmental determinants of a molecular biomarker (DNA methylation) over the course of development in a population of wild spotted hyenas. Similar to many primates, hyenas live in groups organized by a social dominance hierarchy, and whether or not a hyena is born high or low ranking has lifelong consequences.

What difficulties did you run into along the way? 

In this study, we focused on measuring DNA methylation, which is generally of interest to researchers because it is responsive to environmental stimuli and associated with gene expression. Still, while spotted hyenas present a unique opportunity to investigate how various social experiences and ecological factors early in life are associated with biological characteristics later in life, there were no previous studies (at least of which we were aware) that measured DNA methylation in this species. In other words, this was not like working with a well characterized molecular biology model organism, like fruit flies or lab rats. In fact, when we were conducting our lab work there was no publicly available draft hyena genome. In our attempt to assess a potentially informative biomarker in hyenas, we measured multiple types of DNA methylation with varying degrees of success. Finally, the hyenas we study live freely in a large reserve in Kenya, so much of our data were observational and collected under a variety of field conditions making collection of samples non-trivial.

Photo credit: Zach Laubach

What is the biggest or most surprising innovation highlighted in this study? 

This work represents one of a handful of studies conducted in a wild population that measures DNA methylation to better understand how early life environment may influence organisms’ biology over the course of development. Taking advantage of our approximately 30 years’ worth of continuously collected data on individually recognizable hyenas from the Masai Mara Hyena Project, we not only amassed a particularly large sample size for a long-lived, wild mammal, but we were also able to compare patterns of DNA methylation at various stages of development with respect to multiple early life environmental factors. We found that being born to a higher-ranking mom corresponded with greater global DNA methylation in young but not older hyenas. One interpretation of this result is that high ranking moms confer some advantage to their cubs early in life, but that the effect of maternal rank per se is not evident in global DNA methylation of subadult or adult hyenas. We also found some associations between global DNA methylation and litter size, human disturbance, and prey availability in the year a hyena was born, and these associations were strongest in the youngest age group of hyenas.

Moving forward, what are the next steps in this area of research?

In our next steps we are working to understand whether specific types of early life social environments, like maternal care and how well socially connected an animal is within its group, correspond with variation in DNA methylation and adult stress. We are also utilizing more advanced techniques for measuring DNA methylation, so that we might home in on functional pathways that are involved in the development of an adverse stress phenotype. As part of our broader research agenda looking at general biological principles related to DOHaD hypothesis, we have also teamed up with epidemiologists to ask how social status in humans affects biology. In fact, we have recently published another a paper looking at the associations between maternal socioeconomic status and patterns of DNA methylation over the course of development in children who are part of the Project Viva pre-birth cohort study (check out the paper here).

Photo credit: Zach Laubach

What would your message be for students about to start developing or using novel techniques in Molecular Ecology?

This project was part of my PhD work, and from this experience I have learned just how fast molecular biology advances as a field. Given that this technology is constantly changing, it is critical to find mentors and collaborators with up-to-date expertise who are willing to support you. I was fortunate to work in a cutting-edge molecular laboratory, and to receive training from internationally recognized experts in Dr. Dana Dolinoy’s lab who specialize in studying DNA methylation. Additionally, in studies like these that involve large observational data sets and that aim to understand biological mechanisms, the value of clearly defined study questions, hypotheses and a complimentary analytical strategy cannot be understated. In my opinion, novel technology will not substitute for a thoughtful and well-planned analysis.

What have you learned about methods and resources development over the course of this project? 

Working in a novel system, like investigating DNA methylation in wild spotted hyenas, presents challenges and limitations that are unique from those encountered in laboratory settings and when working with model organisms. However, there are deep insights and rich perspective to be gained at the three-way interface between molecular biology, behavioral ecology and evolutionary biology from study populations with intact life histories and that are subject to natural selection. I have also learned that long-term field studies with uninterrupted data collection, like the Masai Mara Hyena Project, provide an invaluable resource and an unmatched opportunity to combine molecular techniques with vast collections of behavioral, demographic and ecological data. In addition, while long-term field studies represent a substantial investment of time and resources, they also present a chance for comparative research that can help elucidate basic biological principals that span taxa, like the DOHaD hypothesis. As such, I believe I have been fortunate to work with Dr. Kay Holekamp’s hyenas and that these types of long-term field studies are an asset to be prioritized and preserved.

Describe the significance of this research for the general scientific community in one sentence.

Social and ecological factors experienced early in life can correspond to changes in molecular biomarkers, like DNA methylation, that are detected over the course of development, and that may affect patterns of gene expression.

Photo credit: Zach Laubach

Describe the significance of this research for your scientific community in one sentence.

Findings from this research suggests that maternal rank, anthropogenic disturbance, and prey availability around the time of birth are associated with later life global DNA methylation in spotted hyenas, particularly in cubs.

Citation: Laubach, ZM, Faulk, CD, Dolinoy, DC, et al. Early life social and ecological determinants of global DNA methylation in wild spotted hyenas. Mol Ecol. 2019; 28: 3799– 3812. https://doi.org/10.1111/mec.15174

National-scale eDNA metabarcoding study reveals diversity patterns of plant pathogens and how they change with land use

Plant pathogens are a major factor in farming and forestry, and also play a key role in ecosystem health. Understanding pathogens at national scales is critical for appropriate prevention and management strategies and for a sustainable provision of future ecosystem services and agroecosystem productivity. Despite this, at present we have little knowledge of the diversity patterns of plant pathogens and how they change with land use at a broad scale.

Photo credit: Ian Dickie

In our study we show how land uses such as farming and plantation forestry affected the variety of plant pathogens in soil, roots and on plant leaves – and we show there are many more species of plant pathogens in land that’s been modified by pasture, cropping, and plantation forestry than there are in natural forest. The patterns of pathogen diversity are distinct from other microbes.

These are some of the first landscape level insights into these critically important communities including fungal, oomycete and bacterial pathogens in seemingly healthy ecosystems. Our results give scientists new insights into where pathogens exist, and how pathogen communities are structured.

Andreas Makiola and Ian Dickie (Bio-Protection Research Centre, New Zealand)

Read the full article here

As genomic and ecological data sets grow larger in size, researchers are flooded with far more information than was available when many conventional model-based approaches were designed. To deal with these massive amounts of data, many researchers have turned to machine learning techniques, which promise the ability to help find signals within the noise of the complex data sets generated by modern sequencing approaches. Applications for machine learning in molecular ecology are broad and include global studies of biodiversity patterns, species delimitation studies, and studies of the genomic architecture of adaptation, among many others. Here at Molecular Ecology Resources, we are excited to highlight research that applies supervised and unsupervised machine learning algorithms to answer questions of interest to the readership of molecular ecology. This special issue will also highlight the nuances and limitations of machine-learning techniques. Rather than focusing on the supposed differences between machine-learning and model-based approaches, this issue would aim to highlight the broad spectrum of machine-learning approaches, many of which can incorporate model-based expectations and predictions.

We are soliciting original research that applies novel robust applications of machine learning methods on molecular data to address questions across ecological disciplines.

Details

Manuscripts should be submitted in the usual way through the Molecular Ecology Resources website. Submissions should clearly state in the cover letter accompanying the submission that you wish the manuscript to be considered for publication as part of this special issue. Pre-submission inquiries are not necessary, but any questions can be directed to: manager.molecol@wiley.com

Special issue editors: Nick Fountain-Jones, Megan Smith & Frédéric Austerlitz

Intra-specific variation and the algal microbiome

Individuals within a species vary, and this variation can have important implications for the role a species may play within ecosystems. We compared the relative importance of variation within species due to genetic changes within its own genome versus symbiotic interactions between the focal species and its associated bacteria, also called their microbiome. We focused on Microcystis aeruginosa, a globally distributed photosynthetic cyanobacterium, also known as blue-green algae, that often dominates freshwater harmful algal blooms.

Colony of Microcystis aeruginosa from Gull Lake. Colony photographed by O. Sarnelle of Michigan State University and image prepared by John Megahan of University of Michigan.

These blooms have recently become more common and intense worldwide, causing major economic and ecological damages. We studied Microcystis and their associated microbiomes from lakes in Michigan, USA that vary in phosphorus content, which is the primary limiting nutrient in lakes. We found genomic changes among strains of Microcystis along this phosphorus gradient that indicated increased efficiency in the use of phosphorus and nitrogen. Intriguingly, we found that genotypes adapted to different nutrient environments co-occurred in phosphorus‐rich lakes. This co-occurrence may have critical implications for understanding how Microcystis blooms persist for many months, long after nutrients become depleted within lakes. Similar to previous findings in for example the human microbiome, we uncovered that the bacteria comprising the microbiomes of Microcystis varied in community composition but were more stable at the level of functional contributions to their hosts across the phosphorus gradient. Finally, while our work was mostly focused on unraveling the genomic underpinnings of nutrient adaptation, we also observed consequences of these differences in Microcystis genome and microbiome composition at a physiological level. In particular, when nutrients were provided in abundance, Microcystis (and its microbiome) that had evolved to thrive in low-phosphorus environments could not grow as rapidly as strains from high-phosphorus environments.

Sara Jackrel, Postdoctoral Fellow, University of Michigan.

Read the full article here.

Citation: Jackrel, SL, White, JD, Evans, JT, et al. Genome evolution and host‐microbiome shifts correspond with intraspecific niche divergence within harmful algal bloom‐forming Microcystis aeruginosaMol Ecol. 2019; 28: 3994– 4011. https://doi.org/10.1111/mec.15198

Interview with the author: Understanding reef bacterial responses to climate change

Climate change is causing dramatic changes to coral reefs and the eukaryotic life they provide habitat for, but what about the bacterial communities? In this interview with the author, Susana Carvalho from the Red Sea Research Center gives us a behind the scenes take on the paper she and colleagues published using autonomous reef monitoring structures (ARMS) to uncover insights into how bacterial communities respond to environmental stress.

See her bio here and the paper here.

An ARMS unit in action.  Photo credits:  Jessica Bouwmeester

What led to your interest in this topic / what was the motivation for this study? 

Coral reefs face unprecedented decline due to local pressures and climate change, making assessment of species diversity and responses to environmental change a priority. Despite the critical roles of bacteria in reef functioning, their communities remain largely overlooked, partially due to the lack of standardized tools and protocols. Fostered by the recently developed Autonomous Reef Monitoring Structures (ARMS), the team – already studying eukaryotes associated with ARMS – decided to expand the research to bacterial communities. This step was further motivated based on the knowledge previously gathered on the eukaryotic reef benthic communities as well as the fact that the Red Sea can be seen as a natural laboratory for ‘Future Oceans’ due to clear environmental gradients in sea surface temperature and salinity. 

What difficulties did you run into along the way? 

The biggest difficulties for this project were logistical and also the fact that it was an exploratory study. Firstly, the reefs which were studied were spread across 2000km of the Saudi Arabian coastline. This posed problems not only in getting permissions from a number of different agencies to visit the reefs but also in organising the logistics so equipment and people could undertake the sampling safely over such a wide spatial extent and across numerous years. Secondly, the nature of the experiment was exploratory, as very little knowledge is known about reefs in the Red Sea. While this makes the experiment vital to further knowledge it also complicates the ability to understand the trends observed as no prior knowledge is available.

What is the biggest or most surprising innovation highlighted in this study? 

While ARMS have previously been used to study the small cryptic eukaryotes (small-sized organisms that are hidden in the reef structure), they had not been utilised to study bacterial communities. For the first time, we employed ARMS to investigate structure and composition of bacterial reef communities across pronounced environmental gradients spanning 16 degrees of latitude. Using this standardized framework, we found that bacterial community structure and diversity aligned with environmental differences. We also found that the decrease in taxonomic diversity was not mirrored by a decrease in functional diversity, suggesting that resilience is not a direct function of taxonomic biodiversity. Importantly, the structure of ARMS devices feature crevices and light-/dark-exposed surfaces as well as exterior and interiors surfaces; in a word it displays many different microhabitats perfectly designed to sample a large diversity of bacteria. This is not possible using other sampling protocols (e.g. water sampling or sediment sampling). The current approach can be expanded to other regions allowing global questions such as the effect of climate change in coral reefs to be addressed and to build a standardized comparable ARMS-based database that allows for meta-analyses beyond the insight from single studies. 

Moving forward, what are the next steps in this area of research?

To fully understand how coral reef microbial communities are being affected by local and global stressors and how the functioning of the reef changes, long term time series are required. Thus, repeated sampling over a number of years is critical. Currently, we have 19 reefs along the Red Sea where ARMS are deployed and retrieved every two-years along with traditional reef surveys (photo-transects for benthos and fish visual censuses). To properly understand the function within the reef we also aim to undertake a metatranscriptomic analysis of the sessile community to see which genes are active. This would ideally be expanded to include manipulative experiments to understand how specific stressors affect not only the community composition but also the functional activity of the biological communities.

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? 

First of all, we would recommend to consider fully the question/issue that they would like to address with these novel techniques. It should be taken in consideration whether the use of molecular tools will benefit the answering of the desired question in comparison with more traditional methods. While novel techniques can bring substantial improvements in understanding questions, such as the current study, sometimes improvements are just marginal, or can just be in cost and speed.

What have you learned about methods and resources development over the course of this project? 

The aim of this project was to characterize the taxonomic and functional diversity of coral reef-associated bacteria using ARMS. Whilst the team had previous experience working with bacterial taxonomic characterization, a ‘deep dive’ had to be undertaken to perform the functional analysis. These areas are rapidly developing with a wealth of new algorithms appearing in the literature. While there is no perfect approach, during the course of this project we learnt that one of the most crucial aspects when developing a new method is to understand both the benefits and the limitations of it. Only by gaining this understanding can you confidentially present your results and highlight the areas in which these techniques can be applied.

Describe the significance of this research for your scientific community in one sentence.

ARMS provide a standardized platform to investigate the response of coral reef-associated bacteria to environmental change, with current results suggesting that this research should be conducted from taxonomic and functional perspectives.

Describe the significance of this research for the general scientific community in one sentence.

This study lays the foundation for a holistic understanding of how reef communities respond to environmental changes and proposes a framework that, if applied worldwide, can be vital in providing answers to global questions such as the impacts of climate change on ecosystem diversity and functioning.

Citation: Pearman, JK, Aylagas, E, Voolstra, CR, Anlauf, H, Villalobos, R, Carvalho, S. Disentangling the complex microbial community of coral reefs using standardized Autonomous Reef Monitoring Structures (ARMS). Mol Ecol. 2019; 28: 3496– 3507. https://doi.org/10.1111/mec.15167

The story behind the article: Climate change impacts on below-ground communities

Free‐air carbon dioxide enrichment (FACE) experiments have dramatically increased our understanding of how plants may respond to future climate change scenarios. These experiments also provide unique opportunities to better predict how below-ground symbiont communities, crucial to plant health, may respond to climate change. Dr Irena Maček and Prof. Alex Dumbrell give us their behind the scenes insights into their paper that combines Illumina HiSeq sequencing and one of the longest running FACE experiment to find novel insights into mycorrhizal fungi communities and climate change.

Check out the full study here.

The Giessen FACE experimental setup. Photo courtesy of Irena Macek

What led to your interest in this topic / what was the motivation for this study?

Arbuscular mycorrhizal (AM) fungi form mutualistic associations with over two‐thirds of plant species, providing numerous benefits in exchange for carbon. Importantly, different AM fungi provide different plant species with different resources. Thus changes to AM fungal communities can alter plant competition and above-ground productivity. This functional differentiation has motivated both of us to explore the ecological mechanisms regulating AM fungal communities, and it was apparent there was a lack of knowledge on how AM fungal communities respond to elevated atmospheric CO2. This is a major research gap, as AM fungi are entirely dependent on their hosts for carbon and changes in photosynthesis in a high CO2 world may influence this. Thus the chance to sample plant roots from one of the longest running free‐air carbon dioxide enrichment (FACE) experiment in the northern hemisphere in Giessen (Gi-FACE) was an excellent opportunity to address this gap in global climate-change research.

What difficulties did you run into along the way? 

Long-term FACE experiments in natural ecosystems are extremely rare, because such set-ups demand stable funding and the persistence of several generations of highly motivated researchers. This would have been a far greater current problem if Prof. Christoph Müller (Justus‐Liebig University Giessen) and his group had not continuously addressed these difficulties since the Gi-FACE experiment’s inception. Another difficulty that we are increasingly finding in metabarcoding research is how to present often extensive and complex data. Dr Dave Clark (University of Essex) was central in working on innovative ways to address this challenge and moving beyond coarse summaries of total community change.

What is the biggest or most surprising innovation highlighted in this study? 

For the first time, we have combined a long-term FACE experiment in a natural habitat with high-throughput molecular sequencing (Illumina HiSeq) and new ways of presenting community data. This has allowed us to see subtle population-level responses within broader community-level responses of AM fungi to elevated atmospheric CO2 across the course of a year.

Moving forward, what are the next steps in this area of research?

Understanding the impacts of global-climate change on terrestrial ecosystems requires an integrative approach that explores responses across all levels of biological organisation and spatiotemporal scales, both above- and below- ground. We still lack a comprehensive understanding about how interactions between the above- and below- ground components of biodiversity respond to both acute short-term (e.g. episodic heatwaves, drought etc.) and chronic longer-term climate changes (e.g. warming, elevated CO2). New experiments aimed at addressing these knowledge gaps with robust levels of replication and appropriate experimental durations for capturing longer or shorter-term responses are required, and these must allow for combined sampling of above- and below- ground biota.

Plant root with arbuscular mycorrhizal fungal structures (hyphae and arbuscules). Photo courtesy of Irena Macek

What would your message be for students about to start developing or using novel techniques in Molecular Ecology? 

The same as our message for all earlier career researchers – identify your research question, read around your research area, develop your hypotheses and plan an appropriate study to address them, and then choose the correct tools/techniques to conduct the research with. The novel techniques can have a lot of analytical power but can also produce a lot of erroneous data due to the rapid development, a lack of testing and a lack of experience. It is very important to initiate the study with clear questions resulting in hypotheses driven research. Do invest time in skills of data analyses and bioinformatics from the very beginning as there is never enough time to do that the later you are in a career, the more difficult it gets with many other obligations.

What have you learned about methods and resources development over the course of this project? 

The field of molecular ecology continuously develops at a rapid pace. It is crucial to have a good network of people with a multidisciplinary range of expertise to collaborate with and to capitalise on all these new and often varied developments. Essentially, good collaboration is crucial. It should also be fun, which is always a good recipe for it to be sustainable and develop into a long-term connection.

Describe the significance of this research for the general scientific community in one sentence.

Elevated levels of atmospheric CO2,reflective of those we will experience in the next ~100yrs, drive changes in symbiotic AM fungal populations with the potential to resonate throughout their associated plant communities, changing above-ground competition dynamics and ecosystem productivity in currently unpredictable ways.

Describe the significance of this research for your scientific community in one sentence.

Predictions regarding future terrestrial ecosystems must consider changes both above-ground and below-ground, but avoid relying on broad‐scale community‐level responses of soil microbes observed on single occasions.

Citation: Maček, I, Clark, DR, Šibanc, N, et al. Impacts of long‐term elevated atmospheric CO2 concentrations on communities of arbuscular mycorrhizal fungi. Mol Ecol. 2019; 28: 3445– 3458. https://doi.org/10.1111/mec.15160Citation: