Environmental RNA and DNA metabarcoding of soil fauna reveal complementary insights into biodiversity and limited effect of nitrification inhibitors

Metabarcoding of environmental DNA (eDNA) is rapidly being adopted to describe and assess biodiversity. However, eDNA has limitations since it captures DNA from both living and dead organisms including relic and extracellular DNA and cannot distinguish metabolically active individuals. In contrast, environmental RNA (eRNA) has the potential to reflect the living organisms. In this study, we compared eRNA and eDNA metabarcoding analysis of soil fauna communities from a cultivated field plot experiment across two seasons to assess the active taxa (eRNA) to the total taxa (eDNA) and hence soil fauna diversity. Both approaches showed distinct community structures, including a large overlap in OTUs shared between the two methods. Seasonal effects were evident in both methods; eDNA detected a higher number of species than eRNA in spring; however, the opposite was observed in fall. Soil fertilizer effect was detected in both approaches, while the effects of nitrification inhibitors (NI) concentration were only significant in the eRNA spring dataset shortly after NI and fertilizer application. This indicates that while eDNA reflects the overall soil fauna diversity, eRNA provides a more sensitive snapshot of the living, transcriptionally active fraction of the community. Our results highlight that eDNA and eRNA metabarcoding of soil fauna provide different but complementary insights into soil biodiversity, creating new opportunities in monitoring soil health and ecosystem functions in soil ecological studies. • eDNA and eRNA metabarcoding produced distinct but overlapping soil fauna communities. • Seasonal diversity patterns differed between eDNA and eRNA datasets. • Nitrification inhibitor concentration effect detected in eRNA in spring