An interview on the Traversing European Coastlines (TREC) project with Yannick Schwab

Traversing European Coastlines (TREC) is a flagship project in the planetary biology theme from European Molecular Biology Laboratory (EMBL). A consortium of several research organisations under the lead of EMBL has embarked on an expedition to sample the biodiversity in the coastlines of Europe in 2023 and 2024. We caught up with Yannick Schwab, Team Leader and Head of Electron Microscopy Core Facility at EMBL, to find out about his group’s involvement in the TREC project. Yannick is using electron microscopy (EM) to describe and analyse the biodiversity of coastal microorganisms and was one of the driving forces behind the advanced mobile lab, which is an innovative tool that is essential for many of the groups involved in the expedition.

What is EMBL TREC (Traversing European Coastlines) project and why is it important?

I’ll start by taking a step back; at EMBL we have indicative schemes where we announce the type of science that we are going to do. They are evaluated by our council and then granted, or not. Our last scheme coincided with the arrival of our new Director General, Edith Heard. She was very keen to develop new themes that we call transversal themes, because they are trying to harness the science from multiple departments. Before that, we had very conventional departments: we had Microbiology labs, Developmental Biology, Cell Biology, Structural Biology, and so on and so forth. We developed various additional transversal themes such as human ecosystems, neuroscience, data science, and so on.

One of the themes is planetary biology, and here the idea is to create an interface between molecular biology, including cell biology, developmental biology, even structural biology, and ecology. Even though a lot of relevant research has been conducted at EMBL for decades, EMBL is not known as a place of an ecology research. We are tackling this by interacting with, and developing strong collaborations with, researchers who are doing ecology. Within this Planetary Ecology theme, there are different research topics and different groups. We have a flagship project that we call TREC, Traversing European Coastlines, which many groups have joined. There’s a big consortium from EMBL, but also way beyond. We are interested in studying the coastline of Europe, and completing a big survey of the biodiversity from microbes to small invertebrates and larger animals at more than 100 coastal sampling sites. So, there’s a big part which is doing an inventory of the biodiversity. Then we want to highlight and study further the impact of gradients on this biodiversity. Gradients can be natural, geographical, temperature and, most interestingly, they can also be anthropogenic. For example, what’s the effect of global warming; what’s the effect of pollutants that are coming into the ecosystems and so on. We are focusing on multiple ecosystems from the land to the sea. In the expedition, we systematically sample organisms in the soil, in the sediments or intertidal zones, in shallow waters, and also further away in the ocean. To do this we have developed strong interactions with multiple marine stations and, very importantly, with the Tara Ocean schooner, which has been surveying microorganisms, plankton, for more than a decade. Then, it becomes a bit more complex because there are more than 150 research teams in nearly 30 countries on TREC, including around 80 plug-in projects. So, it’s more than 150 teams including genomics, transcriptomics, cell biology and interactions in this very complex environment. We are also interested in collecting information about the chemistry in the soils, how pesticides are being metabolised, and by whom. All that is part of TREC (https://www.embl.org/about/info/trec/).

To be able to do our research, we developed mobile laboratories because very often, for multiple techniques, it’s important to have the proper fixation methods to snapshot the living conditions as faithfully as possible. That was the motivation to build a fleet of mobile laboratories. We have a four-wheel drive SUV, which is mostly for bringing material to the sampling sites. We have a sampling van containing equipment that mostly replicates the equipment that they have onboard Tara, to filter plankton. We also have a small van with a miniature lab, where we have a bench, fridges, a small microscope with binoculars, where we can do some quick analysis and conditioning of the samples. These small vehicles are the ones that are travelling throughout TREC, doing 120 transects. Then on top of that, we have built an advanced mobile laboratory. This is a semi-trailer, which is 12.5 metres long and it unfolds to increase the surface area. In total, we have 40 square metres of lab space inside. The trailer is not doing all 120 sites, but it will cover the eight sites that we call the super-sites. The lab will be at each super-site, which are linked to marine stations, for a month or more. The small vehicles and advanced mobile lab travel in parallel, so we always have an overlap.

Can you tell us about your project? Do you access samples from all 120 sites or are you focused on the eight super-sites where you have access to the advanced lab?

Because our work relies on what is inside the advanced mobile lab, we sample only at the 8 super-sites. Our project is focussed on a particular type of microplankton, the dinoflagellates. Our goal is to establish an ultrastructural atlas, cataloguing the diversity of this group. But remember, this is a very minute project amongst others! We want to preserve the ultrastructure as accurately as possible – we want to see how the cells would look if we were looking at them in the sea ­– so we fix them using the high-pressure freezer in the advanced mobile lab. This is the gold standard for fixation. We go out on boats to collect plankton, and then we rush to the harbour and to the marine station. We concentrate the cells and fix them. Typically, we manage to do that within an hour or two of the sample being taken out of the ocean. We did some pilot expeditions to prepare for TREC, and in those we saw a dramatic improvement in the preservation of the ultrastructure when we use the high-pressure freezer. EM has been done on plankton for more than 70 years, but always with chemical fixation. This means that the ultrastructure has never been as optimal as after using the high-pressure freezer.

I have a naive question, why can’t you just bring the samples back to the lab in a bucket of sea water?

As soon as you take them out of the ocean, they start to suffer. They are adapted to their environments and for most of them we don’t know what they need to survive. That’s why many of them cannot be cultured. There’s always one element that is missing and I don’t know what it is, even whether it is a biotic or abiotic factor. As soon as you take them from their environment, they react, and we don’t want that, we want to have a picture of what’s happening in the ecosystem. They don’t like to be concentrated, they don’t like to be handled, so we try to be as protective as possible, but we probably still interfere with them. We concentrate the dinoflagellates by filtering them on a very small mesh size, but this is essential as they are very diluted in the sample.

We mainly use the high-pressure freezer for our project, but inside the truck we have a lot of other equipment, including a plunge freezer, and a cell sorter, which is particularly important. We are currently developing workflows to enable us to purify the plankton. When you collect your buckets of water, it’s a soup, you have plenty of organisms that are completely unique, some of them you can recognise and some of them you don’t. We have groups that are trying to deal with that by systematic metabarcoding. But in some instances, we can recognise a species and here we can be very opportunistic, which is exciting. When we start sampling in a location, we can identify which species are very abundant and we call those the key players. And by developing our workflows around cell sorting, we have managed to purify fractions that contain those key players and then we can prepare them for downstream analysis. Being able to purify many cells opens up all the downstream analysis, including multi-omics, imaging, etc. Here again, while we have a diversity of interests inside the consortium, we all rely on sorting.

Aside from the sample collection, are there lots of collaborations between the different projects?

In our project, we collaborate with a consortium of people who are doing expansion microscopy, which rely on the same fixation methods as for electron microscopy. We collaborate a lot to cross-correlate the results that we are getting. Expansion microscopy is very powerful to reveal the ultrastructure to some extent, but it also brings the ability to label, so you can apply antibodies, or RNA probes, and so on. This is very useful for describing the biology of the cells. So, this is happening now and it’s working very, very well. I think we are setting the ground for many, many discoveries on organisms that are much more diverse compared to the model organisms that we are used to working with.

Are any of the teams doing live cell imaging?

Alongside our work to fix the microorganisms, groups are also trying to cultivate some of them. That has been a bit challenging because we don’t necessarily know many of these species very well. They are taxa that are not usually seen in the labs. This is something that the marine stations and the ecologist partners have already been doing for years, and we do collaborate with them on future collections. They are also helping to bring those new species in to culture. Alongside our cataloguing, we are also trying to interpret our results with the perspective of the presence of chemicals, to correlate them with temperature, with the acidity of the water. It will take years to really unravel all those interactions!

How did you select your super-sites?  Is it because they are quite different in terms of the ecosystems to allow you to complete these kinds of analyses?

It’s a multifactorial decision. Firstly, there needs to be a strong will to collaborate. These marine stations are stakeholders in the TREC expedition and are very motivated to collaborate with us. They are interested in collecting data and integrating molecular biology into their science. They all have long-term records of the biodiversity, which is super important, because TREC, right now, is a one shot. So, we must integrate what we are doing with the time series that they have been collecting. It is also very important that we try to sample across different locations in Europe, and also across gradients. For example, are we close to pristine sites? Or are we close to polluted sites? We already know that sampling at eight sites is not enough, and we know that many other marine stations would love to host the advanced lab. There are already calls for TREC number two!

Do you think that you’ll be able to go around again?

We hope so, yes. We are very motivated to continue.

Do you envision it being a continuous expedition, or would you repeat it again in, say, five years?

Honestly, I don’t know, that is part of the discussion. For TREC itself, I think in terms of the biological project it is very interesting to continue and this could either being more granular, to increase the time series, or to go back to some sites where we have already sampled. And this is just speculative now, but we may highlight changes in biodiversity that are already happening. Then, we can go back to see if it keeps evolving.

On the other hand, we have other countries, groups and institutes that are interested in using the mobile lab. There are a couple of countries that have highlighted their interest in having the mobile lab for a few weeks or months to survey their own ecosystems. So, all these aspects are part of our discussion and we are very motivated by the options. It’s a very exciting thing, because we have essentially reversed the concept of a core facility. Usually, core facilities are hosted in an institute, at a fixed geographical position, and the users come to the facility with their samples. That’s possible for model systems, but as soon as you start to work with wild systems, as I said before, they suffer as soon as you collect them. Here with the mobile labs, we reversed this concept: it’s the facility that is going to the user, to the place where things are happening. I find that super exciting.  With TREC, although the facilities are mainly used for the research that is part of the expedition, local communities are also able to develop projects to use the equipment. So, we already know that this concept of a mobile facility is working and meets existing needs.

Can you tell me about the TREC outreach project?

We have a team for Science Education and Public Engagement that follows TREC. It’s a small team with only two people but they are very active! There is also a strong collaboration with the outreach team at EMBL and those of the visited countries and institutions. I think their work is very important and I would absolutely recommend everyone to visit a site and experience the outreach programme. One of the interactive activities is a big game on a carpet that represents an island, with Pokemon-style cards that represent the biodiversity. In the game, an ‘event’ happens, for example there’s a big spillage of pollutants, and the players have to decide what to do. This is a great way to introduce the need for fundamental knowledge about the species; being able to understand, mechanistically, how they will react and so on. It’s nice to be able to tell them, at the end of the game, that the solutions that they are proposing are what we are doing, just next door in the mobile lab with the researchers. There are also pub talks where researchers from the TREC expedition go to a cinema or a bar and invite the public to come to discuss their work with them, to ask questions. In Bilbao, there were posters everywhere, including in the subway stations!

What were the main research goals from the TREC pilot expedition in Villefranche-sur-Mer bay (France)?

We were developing the workflow for our on-going projects. In our work, we start by selecting a size fraction, for us this means collecting the fraction of 10-40 microns. Although this is removing a lot of things from the plankton, there are still many (100s, 1000s? we do not know yet) different species present. Right now, by EM, it is not possible to look at them all, not in 3D. So, we are developing two approaches, one which is a blind approach, where we create a pellet and prepared it for EM. We cut a 2D section, and we image everything. But in many cases, maybe around 60% of the cases, we don’t know what we are looking at, but, nevertheless, we are doing the analysis, making an inventory of all the organelles that we are seeing. For some species, because of the shape in 2D, we can recognise what they are. For example, dinoflagellates have a very typical nucleus. But aside from the nucleus, they are complex in shape and more than 2000 species have been described so far. So, once you’ve said it’s a dinoflagellate it doesn’t help that much! But still, it’s interesting to start zooming in, and by doing this systematic inventory of organelles we are starting to think that the organelle content is predictive of species. Right now, these are preliminary observations, but for example – and this is trivial – if they have a chloroplast, this is already indicative that they are photosynthetic. In Villefranche, we sampled cells early in the morning, so dark-adapted cells, and then in the afternoon to collect light-adapted cells, and we see changes at the level of the population. There’s a lot of work now to be able to put a tag on the cells at the species level, so this is going to fuel my research for the next few years.

As a next step, as we showed in our latest paper, we can also do very targeted analysis by looking at the cells inside the pellet after they have been prepared for EM. We use fluorescence microscopy, and also transmitted light, to see the global shape of the cells and by looking at the autofluorescence, we can also tell if they contain a chloroplast or not. That helps a lot to narrow down where we need to look by EM, identifying a particular species of dinoflagellate by its shape. This means we can go to much lower-throughput methods, yet powerful in resolution, such as FIB-SEM (focused-ion beam scanning electron microscopy) to do analysis of particular species. In the paper, we focus on one cell type, but we have ongoing work on a handful of different species that we are systematically analysing. So, without spoiling too much, what strikes me the most is that these cells are super-stereotypic in their subcellular morphology. I mean, they are already very stereotypic in their extracellular shape, that is how they have been described for almost two centuries. But if we look inside, we find that the organelles are not randomly positioned. There’s a very specific morphogenesis that is happening and I am very interested in understanding it better.

Right now, in TREC, we are collecting samples, and the analysis will take years. I know I will not have the capacity to study everything, so we also use TREC as an opportunity to build a biobank. So, when my team collects samples for our own projects, we collect additional samples that we keep in liquid nitrogen and also embed in plastic. We want to make these collections openly accessible. People will have access to the metadata that we are collecting alongside, and we will do metabarcoding so that we have an idea of the species that are present in our blocks. The resin embedded blocks will also be systematically imaged with X-rays, so we can have an idea of what is inside. My dream is to make this resource available to the international scientific community for future research.

This sounds like an amazing resource, but will storage start to become an issue?

We have built a central hub here as part of TREC with the capacity to store the samples, not only for imaging but also for genomics.

Does the money for TREC come from EMBL?

TREC is part of EMBL’s current five-year scientific programme ‘Molecules to Ecosystems’, and is funded through the contributions of EMBL’s 28 member states. Our partners are also contributing financially and through in-kind support, and we are also receiving support from external donors.. Philanthropic donations were especially crucial to build the mobile laboratories. We have also benefited from European grants, including support from a big European grant called BIOcean5D.

Was there anything that was more challenging than you expected, or conversely anything that went more smoothly?

I think that the big challenge is coming next. I don’t think we fully comprehend the complexity of analysis of all the data that we are collecting. Otherwise, I think all the logistics, the buildup of the mobile lab and so on, have been a lot of work upstream. But we had an amazing group of people thinking ahead and preparing all that, which I think made it run pretty smoothly. On the scientific side, we have very experienced groups that are coming in to do their research, and we had pilot expeditions that helped us prepare. The mobile lab is the first of its kind. So here, I think, we can be pretty proud because it is working very well. That was a bit of a challenge because of the time pressure, dealing with manufacturers and issues with the supply chains, which meant that we were not able to have the semi-trailer ready when the TREC expedition started. So, for the first couple of stops, we actually had to ship the equipment from one site to the next. But starting at a super-site in Sweden, we have had the mobile lab, and honestly, from day one, it was working.

How long did it take to plan and fit the semi-trailer?

We first made contact with the truck company to discuss the concept and to try and establish a quote in February 2022. Then, we kick started the real work. So, we placed the order around June and then there was a very intense preparation phase, where we discussed with engineers from the company. We drafted plans; where to place all the plugs and so on. That lasted until January. And then the first piece of the trailer was delivered to the factory in February 2023. It took them a few months, until the end of July, to finish the truck. Then it was delivered to us in early August, in Sweden, and the next day it was working!

Is there anything else that you’re excited about in the TREC project that we haven’t discussed?

So, I have focused on my work, but on the imaging side, I’m very excited about the research of our collaborators. I mentioned the plunge freezing together with the high pressure freezing, which is also used to prepare samples for expansion microscopy. This is going to be absolutely insane: identifying the molecular players of the different marine microorganisms that we are looking at. But also very excitingly, we have structural biologists on-board. They are using the plunge freezer to prepare cells for electron cryo-tomography. And I want to believe that we are starting, or at least enhancing, a new field of environmental structural biology. For me, this has huge potential. People are interested in molecular machines and they are used to studying them from inside out in model systems in the labs. But now with the mobile lab, we are opening this up to wild specimens. And I’m pretty sure that we’re going to be able to see biodiversity at the molecular level, with atomic resolution. I find that completely mind blowing, even though I’m not a structural biologist myself. I see the potential being enormous. I think the latest technologies in this field, which are more precise and with higher resolution, are ripe to go for crazy projects like this! This field shows the limitation of possibilities we experience in our conventionl lab system. However, if you consider the overall tree of life, we are looking only at a very tiny corner of it in the labs. And I think it is urgent that we do it now because of the massive collapse of biodiversity – we need to collect as much as possible while we still have time.

My final question is, when you first started on this project, did you think it was a crazy idea, or did you think that it was quite doable?

No, I was excited, but I always thought it was quite doable. Bringing EM equipment to the mobile lab: this stemmed from an ongoing collaboration that I have with Johan Decelle. Johan is studying symbiotic specimens that are impossible to cultivate. He was collecting them in Villefranche-sur-Mer and then drove them to Grenoble to high-pressure freeze them. To improve this workflow, we had the idea of bringing a high-pressure freezer on to the beach, and fortunately Leica supported us to do that. While we were working on this project, the TREC theme of Planetary Biology was born, including the idea of a mobile laboratory. Because we already had these kinds of ideas in our project with Johan, we said, let’s try to bring advanced imaging inside the mobile laboratory. It was serendipitous somehow, but a perfect alignment of the planets in a good moment and a good place, and it does work! I think sampling is relatively easy because we also benefit from the experience of marine stations, which have been sampling marine organisms for many years. And of course, we are very familiar with our own instruments – although we didn’t know if they would work in the trailer. And finally, we were working with specialists who know how to customize a trailer. This meant that the sampling part of the project was all about bundling expertise and not too much of a risk. I think the challenge is ahead of us: how are we going to make sense of what we are seeing? This is not a reductionist approach; we have the full complexity of ecosystems to understand and now we are trying to make sense of what we are looking at!

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