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Ants serve as ecosystem engineers that maintain important ecological processes within forests. Given their ecological importance, it is a clear scientific shortcoming that we lack non-invasive methods to survey their behaviour inside common opaque habitats such as mounds, litter, and soil. In this study, we assess if acoustic signals from red wood ant (Formica rufa) mounds are useful to infer temporal changes in ant activity within forested ecosystems. We found that acoustic indices used previously as a proxy for soil fauna in soil ecological studies (Acoustic Complexity Index, Bioacoustic Index) can indeed separate sounds generated by the ant's daily routines (biophony) from other forest sounds. Yet, we also show that these indices are problematic proxies for soil diversity as they increase not only due to an increased number of species but also due to an increased number of the same species. Acoustic measures that incorporated the strength of acoustic signals, Average Power Density (APD) and Peak Power Density (PPD) also increased with increasing ant abundance and constituted the conceptually best proxy for ant activity. For example, the PPD could i) track diurnal changes in Formica rufa activity with a high temporal resolution (minutes) and ii) detect altered behavioural responses to temperature changes. We conclude that microphones detecting biophony can provide high-resolution information about in situ ant behaviours in forested ecosystems. Thus, passive acoustics monitoring offers a promising avenue as a non-invasive monitoring tool for soil macrofauna studies.

Soil-dwelling insects may be at particularly high risk of exposure to pesticides because soils are a major recipient of these chemicals. Still, we lack non-invasive techniques to detect in situ responses of soil macrofauna to pesticides and other contaminants. Here, we test if sound recordings can reveal sub-lethal behavioural shifts in ant colonies caused by a neonicotinoid (imidacloprid). We demonstrate that an acoustic variable can detect pesticide-induced shifts in outdoor behaviours (increased activity) of red wood ants (Formica rufa). This behaviour shift was observed at environmentally relevant, sub-lethal levels of imidacloprid (10.8 ± 5.7 ng g-1 sample, average ± standard error). Notably, our recordings revealed a short-term (<6 h) increase in activity induced by the pesticide, followed by a slow recovery and a return to the baseline over a weekly timescale. The long-term sub-lethal behavioural effects would have been difficult to detect using other techniques, given the opaque nature of the studied mounds. We conclude that acoustic monitoring offers a promising tool for detecting pesticide effects in natural settings. We also provide recommendations for best practices for acoustic monitoring of soil fauna and discuss the challenges of measuring soil fauna behavioural changes in a natural setting.