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 density of life 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 water 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 or 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, including the implementation of monitoring and assessment of conservation measures and the minimization of 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 vehicle of heredity and the hallmark of living organisms (as opposed to water, rocks, etc.), it can be extracted from the environment.
Indeed, living beings leave traces of their presence in their environment: tiny droplets of saliva, hair, mucus, skin, scales, feces, decomposing cells… These DNA-laden remnants float in the air, carried by air currents, and settle on the ground or in 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.
Uwe Kils/Wikipedia, CC BY-SA
We are now able to identify specific fragments within genome sequences or mixtures of genomes (such as in DNA) that vary sufficiently across 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, across space and time
In microbiology, such 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 broadly speaking, the ability of DNA-based approaches to detect and distinguish cryptic species (which cannot be identified based on morphological criteria) has been harnessed in a wide variety of environments. Thus,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 more plankton lineages than 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 of microalgal communities following World War II and the introduction of agricultural inputs, while analysis of contemporary insect communities in pine forests reflects the state of habitat decline in the face of climate variations.
An implementation process that is both simple and careful
To begin with, one must filter the air or water, or collect soil or sediment, but this relatively simple process requires extreme caution, as the DNA contained in these samples is often present in very small quantities. At the bottom of the ocean, life is rare, even if 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 the workstations of ships. It is often even rarer in seawater, where it degrades very quickly after cell death. 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 being carefully packaged to prevent contamination and transported to the laboratory, the samples undergo environmental DNA extraction using various methods (chemical, mechanical, or filtration), depending on the objective and the environmental substrate—whether it be freshwater, seawater, sediment, soil, etc. Once the DNA is 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 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—following the approach taken in Tara Ocean—we have developed a dual-pronged approach to characterize both unicellular prokaryotes (bacteria and archaea) and eukaryotes (unicellular protists, animals, fungi, etc.). All organisms can be studied by constructing “metabarcode libraries” targeting small genome fragments that allow species or lineages to be distinguished from one another; 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 lives in sedimentary substrates) and pelagic fauna (which lives 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 selects the barcodes that reveal diversity across the tree of life, from bacteria to animals.
These protocols, implemented by the “eDNAbyss” project, have generated 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 integrate 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 Ocean. The results highlighted several key insights into our understanding of marine biodiversity distribution. They revealed a level of diversity three times higher in benthic sediment communities compared to pelagic communities in the water column. Among 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.
Comparing plankton species found at the surface with those found in the sediment also helpedidentify key players in the biological carbon cycle, a process critical to the climate.
This first step is an encouraging demonstration of the potential 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.