RES mitigation banks in Georgia are supporting more macroinvertebrate diversity than traditional net sampling was capturing. Environmental DNA metabarcoding is how we found out — and it points to a more complete approach to monitoring restoration effectiveness.
Macroinvertebrate communities are a reliable indicator of stream health. The variety of aquatic insects living in a channel, which species are present, in what numbers, and across which functional groups, tells ecologists a great deal about whether a restored stream is functioning the way it should. Getting an accurate read on that community, however, depends heavily on how you sample it.
Environmental DNA (eDNA) has been used for over two decades to non-invasively detect organisms in their environment. More recently, the increased availability of next-generation DNA sequencing and advanced bioinformatics has allowed scientists to describe whole communities of organisms from the genetic material they shed into the environment — an approach known as eDNA metabarcoding. Scientists extract and analyze that genetic material and use it to estimate the number and types of organisms present at a given location. Applied to stream macroinvertebrates, it offers a way to detect insects that traditional net surveys struggle to reach.
A net survey works by disturbing the streambed or banks and capturing what drifts into the net. It's a well-established method with a long regulatory track record, and it performs well for insects that live in open gravel or cling to material in the current — the Ephemeroptera, Plecoptera, and Trichoptera (mayflies, stoneflies, and caddisflies) that most biological assessment indices are built around.
Perlids (Perlidae) are a family of stoneflies (Order Plecoptera) that have large predatory larvae, which are indicators of good water quality in streams.
Some groups of macroinvertebrates are harder to sample depending on where they live. Burrowing insects spend most of their time buried in fine sediment rather than in the water column. Shredders, which process leaf litter and woody debris, tend to be associated with organic matter in snags or at the stream margins. Because they occupy these hard-to-sample habitats, net surveys may chronically underrepresent these insects —not because they aren't there, but because they are more difficult to sample.
Water flowing through organic matter and sediment collects the DNA of the organisms it passes, making eDNA an effective tool for sampling in these habitats. This integrated biodiversity signal allows ecologists to survey a larger section of stream with fewer samples and less disturbance to the environments they are working to restore.
Dobsonflies (Corydalinae) have large aquatic larvae, commonly called hellgrammites, which are abundant across a variety of stream conditions.
Every organism living in a stream continuously sheds genetic material into the water (cells, mucus, waste). To sample eDNA, scientists collect a water sample, extract the genetic material, read the DNA sequences, and match them against molecular databases to identify which species or taxa were recently present in that reach.
RES has been developing and applying eDNA methods across its mitigation bank portfolio, building on fish species detection work at the Mule Train Stream and Wetland Mitigation Bank in Tennessee and on foundational research on eDNA behavior in river systems conducted during the Klamath River restoration. The macroinvertebrate protocol was adapted from that Klamath work—same method, different primer. While the Klamath protocol targeted fish DNA, RES selected a primer optimized for aquatic invertebrate detection based on a meta-analysis of available options and then built the monitoring workflow around it.
The team ran paired eDNA and net surveys at the Blue Creek and Mulberry mitigation banks and at reference streams over two years. The results were consistent across sites and monitoring periods.
Overall, eDNA detected more taxa than the net survey protocol — a finding that held across functional groups and sites. For burrowers specifically, eDNA detected roughly twice the taxa that net surveys found at Blue Creek. For shredders, the difference was more pronounced: eDNA detected approximately seven times as many shredder taxa as the net surveys at the same site. The pattern held at the reference site as well, with eDNA consistently returning higher counts for both groups.
eDNA vs. net survey detection of shredders and burrowers at Blue Creek, Mulberry, and a reference site.
Net surveys performed well for clingers and EPT taxa, consistent with expectations given where those insects live. The two methods detect many of the same parts of the macroinvertebrate community but differ in others. That overlap is informative in itself: understanding where the methods agree and where they diverge will help guide the integration of eDNA sampling into the restoration monitoring process going forward.
Macroinvertebrate diversity scores — including the representation of shredders, burrowers, and EPT taxa — factor into annual monitoring reports at stream mitigation banks. When those scores fall below performance thresholds, the U.S. Army Corps of Engineers withholds credit releases pending further review or for additional monitoring cycles. If a stream is performing ecologically, but the net survey didn't fully capture what was present, the result is a sampling problem rather than an ecological one, and that's where a more complete detection method has direct value.
eDNA is not yet an approved standalone monitoring method for the biological performance standards of mitigation banks. RES is running it in parallel with required net sampling and working to further improve the tool, with the goal of formalizing acceptance at the regulatory level. The multi-year paired dataset collected at the Georgia sites is central to that effort, demonstrating year-over-year consistency and detection performance across multiple sites as the protocol continues to develop.
The current work focuses on closing the remaining detection gaps in the molecular reference database. When an insect’s DNA has never been sequenced and published, eDNA analysis can’t identify it—not because it isn’t there, but because there’s nothing to match it against. The RES team is identifying which taxa appear in net samples but not in eDNA results, with the goal of sequencing those organisms and contributing them to the reference databases, so that future sampling captures a more complete picture.
As library coverage improves and the paired dataset grows, the case for eDNA as a regulatory monitoring method will strengthen. Integrating traditional net sampling and metabarcoding into restoration monitoring is the path forward—one that gives ecologists, bank sponsors, and regulators a more accurate and replicable read on stream biological performance.
Contact our team to talk through how eDNA applies to your site.