Pollinators provide essential ecosystem services and are critical components of resilient ecosystems. Yet, pollinators are suffering staggering global declines leading to increased listing under the Endangered Species Act. This presents challenges for United States Department of Defense installations. Range closures must occur to facilitate surveys, but conventional sampling is costly, time consuming, and increases range closure time, which erodes mission readiness and decreases lethality. Rapid, efficient, and accurate assessment of pollinator communities is therefore essential to maximize return-on-investment. Here, the project team assessed the potential for environmental DNA (eDNA) metabarcoding as a means to maximize return on investment. Specifically, the project team tested if eDNA metabarcoding could provide a viable method of documenting pollinator communities.

The technical objectives were to: 1) develop and validate broad-spectrum pollinator eDNA metabarcoding primers, 2) test the efficacy of multiple capture methods to obtain Next Generation Sequencing-ready pollinator eDNA from flowers, and 3) validate eDNA sampling and microfluidic metabarcoding methods in controlled and field experiments with mixed plant/pollinator communities.

The project team conducted in silico and in vitro testing on a broad spectrum of arthropod metabarcoding primers. They established a greenhouse flower community, introduced a colony of common eastern bumblebee (Bombus impatiens) pollinators, and obtained eDNA samples from four focal flower species (Monarda, Penstemon, Solanum, Cynoglossum) via three sampling methods (nectar draws, flower swabs, whole flower heads). Samples were preserved and extracted using two methods, Phenol-Chloroform-Isoamyl and Qiagen DNeasy Blood and Tissue kit, before being subjected to microfluidic eDNA metabarcoding using the validated primer panel. Sequence reads were analyzed with two bioinformatics pipelines for quality and length filtering, merging, dereplication, and taxonomic assignment.

In silico and in vitro testing revealed a panel of 15 primer sets that showed promise for amplifying pollinator DNA. Microfluidic eDNA metabarcoding detected not only the focal taxon, but also beneficial insect species released historically in the greenhouse as well as common arthropods known to occur in the region. In addition, field samples provided preliminary support for ex situ validation of methods.

The study demonstrated three significant benefits of microfluidic eDNA metabarcoding of pollinator communities: 1) eDNA metabarcoding reduced both time and effort needed to acquire the same level of information and the financial burden of conventional surveys, 2) eDNA metabarcoding has the potential to detect federally listed species, 3) eDNA sampling requires considerably less time in the field compared to traditional methodologies, minimizing range closure time and increasing mission readiness, and 4) eDNA metabarcoding approaches can reveal complex networks that undergird critical ecosystem functions and services, ultimately allowing for an assessment of ecosystem resilience.