Exploring the Deep Sea Through Environmental DNA, or How to Uncover Unexpected Biodiversity
The ocean covers two-thirds of the planet, more than half of which lies at depths of over 3,000 meters. We know very little about the biodiversity that inhabits the deep sea.
Sophie Arnaud Haond, University of Montpellier
Given the low biomass density in these environments, describing species or conducting biodiversity surveys requires handling large quantities of sediment that must be brought up through thousands of meters of the water column and sorted for weeks in the laboratory. As a result, describing a single species or surveying the biodiversity contained in just a few cubic centimeters of sediment can take weeks.
Uwe Kils/Wikipedia, CC BY-SA
However, while the deep sea may be out of sight, it is not immune to human impacts such as pollution (agricultural inputs, maritime traffic, etc.) or the direct impact of the exploitation of the many resources it contains (oil drilling, fishing, etc.). Among deep-sea exploitation projects, the extraction of energy and mineral resources—such as the Pacific nodule fields—is becoming increasingly advanced.
Gaining a better understanding of the extent of marine biodiversity is becoming a major challenge—both for our knowledge of living organisms and their evolution, and for conservation efforts, as well as for establishing monitoring and assessment measures to conserve marine life and minimize environmental impacts. It is in this context that Ifremer launched the “Pourquoi Pas les Abysses” project, which was followed by the France Génomique project “eDNAbyss”: both rely on the use of what is knownas “environmental DNA,” or eDNA.
What is environmental DNA?
Although DNA is the carrier of heredity and the hallmark of living organisms (as opposed to water, rocks, etc.), it can be extracted from the environment.
In fact, living beings leave traces of their presence in their environment: microdroplets of saliva, hair, mucus, skin, scales, feces, decomposing cells… These DNA-rich remnants float through the air, carried by air currents, and settle in the soil or sediment. Thus, much like forensic investigators analyzing suspect DNA at crime scenes, biologists extract DNA from environmental samples to identify the species that live there or have passed through the area.
Uwe Kils/Wikipedia, CC BY-SA
We are now able to identify specific fragments in genome extracts or mixtures of genomes (such as in DNA) that vary sufficiently between species so that their sequences can be used to identify the major groups to which they belong (species, genera, families, etc.). By analogy with barcodes used in commerce, these fragments are called “ barcodes.”
The discovery of unexplored aspects of biodiversity, both in space and time
In microbiology, these kinds of advances have had a profound impact, since the vast majority of microbial organisms—bacteria and archaea—are uncultivable: their characterization has been possible only through DNA analysis. The tree of life has been enriched with a large number of major lineages, notablyarchaea that inhabit hot springs and the ocean floor, and which are the subject of fundamental hypotheses for understanding the ancestral diversification of life into three kingdoms (bacteria, archaea, and eukaryotes).
In ecology in the broadest sense, the ability of DNA-based approaches to detect and distinguish cryptic species (those that cannot be identified based on morphological criteria) has been harnessed in a wide variety of environments. For example,the “Tara Ocean” expedition, by combining morphological and DNA analyses, revolutionized our understanding of plankton diversity in the oceans by revealing the existence of nearly fifteen times as many plankton lineages as the 11,000 previously described.
Beyond what is invisible or inaccessible in space, analyzing the DNA contained in sediments also makes it possible to reconstruct past communities and infer the impact of local or global changes. Analyses conducted in Brest Harbor have shown the upheaval in microalgal communities following World War II and the introduction of agricultural inputs, while analysis of contemporary insect communities in pine forests reflects the decline of the habitat in the face of climate variations.
An implementation process that is both simple and careful
To begin with, you have to filter the air or water, or collect soil or sediment, but this relatively simple process requires extreme caution, since the DNA contained in these samples is often present in very small quantities. At the bottom of the ocean, life is rare, even though it is highly diverse, and the DNA contained in a handful of sediment is present in small quantities compared to that carried by researchers or naturally accumulated on workstations aboard ships. It is often even rarer in seawater, where it degrades very quickly after cells die. It is therefore essential to protect it from potential contamination from sources with much higher DNA content, such as the hands or saliva of those handling the samples, for example.
After carefully packaging the samples to prevent contamination and transporting them to the laboratory, environmental DNA is extracted using various methods (chemical, mechanical, filtration), depending on the objective and the environmental substrate—whether it is freshwater, seawater, sediment, soil, etc. Once the DNA has been extracted, it is used to create “libraries” of various types, depending on the scientific objective and the living organisms being targeted.
The Deep Environment: The Unknown
The marine environment, in all three dimensions, is vast: it accounts for more than 95% of the Earth’s biome. Gaining a comprehensive understanding of the diversity it contains requires standardized research methods across its various compartments and ecosystems. One of the benefits of DNA-based approaches is that they allow the scientific community to pool the results obtained across a variety of ecosystems, like a giant puzzle that takes shape as pieces are added.
Daniel Leduc, World Register of Marine Species, CC BY-NC-SA
To this end, as part of the “Pourquoi Pas les Abysses” project—and as had been done in the Tara Ocean project—we developed a two-pronged approach to characterize both unicellular prokaryotes (bacteria and archaea) and eukaryotes (unicellular protists, animals, fungi, etc.). All of these organisms can be studied by constructing “metabarcode libraries” that target small genome fragments, allowing us to distinguish between species or lineages; and to refine the identification of prokaryotes, their complete genomes are reconstructed using “metagenome libraries.”
The metabarcoding project required a three-step pilot study. The first step involved selecting different sampling methods for benthic fauna (which live on the sedimentary seafloor) and pelagic fauna (which live in the water column). Since DNA can be preserved for long periods in sediment, the next step was to select an extraction method that would facilitate surveys of contemporary biodiversity. Finally, a set of molecular probes identifies the barcodes that reveal diversity across the tree of life, from bacteria to animals.
These protocols, implemented by the “eDNAbyss” project, have made it possible to generate billions of sequences from samples collected from the Mediterranean Sea to the Pacific Ocean, at depths ranging from 300 to 10,000 meters.
C. Schulze and A. Schmidt-Rhaesa, World Register of Marine Species, CC BY-NC-SA
Based on the initial results, it was possible to combine benthic biodiversity data (associated with sediment)—including data generated by the “Pourquoi pas les Abysses?” and “eDNAbyss” projects—with the DNA-based inventories of biodiversity in the water column conducted by Tara Océan. The results highlighted several key insights into our understanding of the distribution of marine biodiversity. They revealed that benthic communities in the sediments are three times more diverse than pelagic communities in the water column. Of this vast diversity on the seafloor, more than a third remains completely unknown: the barcodes do not match any described species included in the reference databases.
A comparison of plankton species found at the surface and in the sediment also helpedidentify major contributors to the biological carbon pump, a key process for the climate.
This first step is an encouraging demonstration of the ability of environmental DNA-based approaches to enable not only standardized and interoperable inventories of biodiversity across the three dimensions of the ocean, but also a better understanding of the major processes to which they contribute. And the majority of the results are still being analyzed…
Sophie Arnaud Haond, Researcher, University of Montpellier
This article is republished from The Conversation under a Creative Commons license. Readthe original article.