Research projects often apply single-cell technologies to differentiate cell subtypes harvested from a pluricellular organism. However, those technologies are also well-suited to characterize and identify unknown unicellular eukaryotes and prokaryotes and shed light on the composition of environmental samples. Engineer Sarah Romac, at the department of Adaptation and Diversity in the Marine Environment (CNRS and Sorbonne Université, France), shares with us their ongoing efforts to describe new species of zooplankton.
Single-cell resolution technologies are critical for non-culturable plankton
Sarah Romac: “We have been collecting and characterizing samples from oceanographic expeditions for many years, but in the plankton phylogenetic tree there are still taxa with very little morpho-genetic data. Organisms from these taxa have not survived collection, fixation, transport, or culture in the lab. What we are trying to do is to overcome those issues to complete this tree.”
“Our main issue working with protists is the inability to grow them in a lab. One of my PIs works with radiolarians, which are unicellular eukaryotes unable to perform photosynthesis by themselves. They require specific microalgae to survive in the ocean, and we cannot reproduce satisfying conditions in the lab. This is where single-cell technologies come in.”
“On an oceanographic expedition, you can harvest your samples, store them, and perform morphological observations, but molecular biology is often nearly impossible due to space and structural issues. I had to set up the entire methodology for processing samples to obtain all the meta-omics, meaning meta-transcriptomics, meta-genomics and meta-barcoding data, from the boat to the sequencing platform.”
“We harvest our samples, store them, bring them back and try to grow them in the lab. It is often from the lab cultures of microalgae that we can characterize new species, morphologically under the microscope, either optical or electronic, and according to their genetic material.”
“By expanding our databases, we can use biomarkers to assign our sequences and identify the species we have in a sample. But there are many sequences that do not find a match and remain unassigned. To solve this issue, our only approach to identify those unknown sequences is to go for single-cell resolution studies. You need to return to the field sample and manually pick individual cells you’d like to characterize, and extract as much information as you can since you cannot grow them in a culture.”
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High biodiversity in environmental samples require a multi-step approach
SR: “Everything is still done manually, depending for example on the size of the microorganisms. I work on species larger than 40µm: we can’t use flow cytometry, we’re doing it the old-fashioned way, under the microscope, with a pipette and sucking out the cells one by one. It is patient work, and it can take a while searching for a specific organism in a sample.”
“We start with organisms we expect to be difficult to cultivate. Either alive or fixed, we take their pictures and extract their genetic information. We usually find sequences that are unknown to our databases, but whose relative abundances can be correlated. We find patterns with different sequences that are very highly present in the same environmental samples. Other patterns we recognize, but we do not understand why their abundances correlate. It might highlight relationships, potentially symbiotic, between different species.”
“We then return to the initial fixed sample, or to the collection site, and we use microscopes to individually target the cells from which one of the sequences in such patterns belongs. We then perform further genomic approaches, either by cloning or meta-barcoding, and push our observations a bit further with microscopy, like highlighting cellular organelles. This enables us to highlight symbioses not yet known in plankton.”
“Until now, single-cell meta-omics projects have been a bit of a misnomer: we usually use a few cells of the same species coupled together, not just one, or single very large cells. We also pre-amplify the genome or transcriptome for meta-omics using whole-genome amplification (WGA) or Multiple displacement amplification (MDA). For radiolarians, we are now able to build a transcriptome from a single cell.”
The quest for a universal method of plankton data extraction
SR: “When working with environmental samples, there are many parameters influencing your results. For example, you are already skewing your data during collection depending on the fixation method you have decided on. If you choose between fixatives such as formaldehyde or Lugol, then you won’t end up with the same species in the final sample.”
“In the past, we had a very specific method for each type of cell. I am currently trying to develop a standardized method that can be applied to all taxa to recover in one go DNA, RNA, and information about the cell skeleton to make a morphological characterization. Sort of a complete description of the species but applicable to all and comparable from taxon to taxon.”
“It is all at an exploratory phase at the moment. For instance, I had developed a new lysis protocol mainly based on mechanical stress, drastic enough to recover as many cells as possible in the sample and avoiding specific chemical- and enzymatic- based degradations.”
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An unusual technical hurdle for single-cell studies: a plankton’s shell
SR: “The most delicate step in our single-cell protocol is to succeed in lysing the membranes but without damaging the cell skeleton to allow a morphological description. We take a picture with an optical microscope, down to a 3-5µm resolution, then we lyse the cell to extract only the nuclear material. Finally, we analyze the skeleton, which must be kept intact, using a scanning electron microscope. For example, some radiolarian shells have a silica-based structure, others are calcium-based, and it is important for species characterization.”
“To develop efficient methodologies, we start with cultured samples, as positive control and for benchmarking, and on fresh environmental samples. The station we work at [Roscoff, France] is by the shore so it is easy, but otherwise we go for another expedition.”
“Once we are confident, then we come back to fixed samples from older expeditions, like the TARA Oceans expeditions, to produce more data using single cells.”
The future of the single-plankton characterization pipeline
SR: Of course, it would be interesting to include proteomics and metabolomics in our research. But we are progressing one step at a time, focusing now on successfully extracting DNA and RNA consistently. First, we would like to see if from very small amounts of genetic material we can reconstruct genomes and transcriptomes from our single-cell samples. Then we can think about expanding our methods. We have enough work for 3 to 5 years! We are also looking for ways to automate the process whenever possible, while accommodating the huge diversity in the taxa of zooplankton.”