Genetic Indicator Research
In the past, data for biological indicator species, such as benthic organisms, phytoplankton, zooplankton, came entirely from morphological enumeration. This means that the samples were examined under a microscope by trained taxonomists who used physical features (shape, color patterns, presence of spines) to determine what kinds of organisms were present. It is now possible to identify the organisms present at a site based on analysis of their DNA.
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Collecting DNA
EPA researchers and collaborators collect and use DNA to help determine species present. There are two distinct ways that such DNA can be obtained. One is to collect the organisms just as one would for morphological enumeration (using zooplankton nets, for example) and then extracting DNA from their tissues. The other is to collect water or sediment samples and from them extract DNA that organisms have shed into the environment (via bodily waste, sloughed skin, released sperm, etc.) which is termed “eDNA” (the leading “e” standing for “environmental”).
The use of eDNA offers some advantages over having to collect the organisms themselves. It can be easy to find in the environment and is rapidly and non-invasively obtained. A disadvantage of using eDNA is the difficulty in confirming if a species was living in a certain aquatic system. For example, was that fish really in the lake or did its DNA wash in from the fish market upstream?
DNA-Based Species Determination
EPA researchers use two common approaches to DNA-based species determination. One is the target marker approach, which uses a chemical probe designed to bind only with the DNA of predetermined species of concern (for example Asian Carp threatening to invade the Great Lakes via Chicago area waterways).
The other is the metabarcoding approach, where the spectrum of DNA sequences present in a sample is determined and then assigned to species identities. This is done by matching the identities to reference sequences catalogued in DNA barcode libraries.
The metabarcoding approach is more relevant to generating biological indicators because it produces a list of all the organisms present within broad taxonomic groups (not just a handful of target species). However, metabarcoding is analytically much more complicated, which is why it remains an area of active research and development.
Read EPA's publications on the metabarcoding approach in watersheds.
Research Areas
On the metabarcoding side, EPA works on the refinement of the environmental DNA extraction and sequencing process, and on the expansion of the barcode libraries. This work incorporates genetics and taxonomy science.
On the biological indicator development side, EPA teams work to better understand how species lists obtained from DNA data can substitute for lists obtained from morphological enumeration. These two lists are conceptually similar, but DNA data does not offer the same quantitative information (e.g., it does not count number of organisms from a species).
EPA teams also work to develop DNA-based indicators for groups such as microbes, whose diversity in the environment remains poorly understood because only a subset is amenable to laboratory culturing.
Finally, EPA teams work on how DNA sample collection can be incorporated into National Aquatic Research Survey (NARS) field routine, for example to accommodate the need for additional sample handling and preservation techniques.