Identifying key parameters for reliable assessment of entomopathogenic nematodes viability as affected by spray application stress‐related factors

BACKGROUND: Conventional pesticide application equipment (PAE) is used to apply entomopathogenic nematode (EPN)-based bioinsecticides, but their closed hydraulic systems could raise the temperature of the spray mixture up to 40 °C, potentially harming EPN, since temperatures above 30 °C can immobilize nematodes, reducing their infective capacity. This study aimed to identify the most suitable method to evaluate EPN viability under the effects of PAE technology. RESULTS: Three EPN species—Heterorhabditis bacteriophora, Steinernema feltiae, and Steinernema carpocapsae—were exposed to thermal stress (10, 20, 30, and 40 °C for 270 min) to simulate spray application conditions. Three viability evaluation methods were compared: prodding stimulation, NaCl chemical stimulation, and no stimulation. Viability was measured by two parameters depending on the assessment method: % actively EPN moving (activity), or % total live EPN, both actively moving and immobile (survival). Additionally, a novel parameter estimating non-lethal stress (Δnl s) was defined by measuring the live but inactive EPNs. NaCl stimulation was optimized comparing different concentrations and durations and then set at 0.1 g mL−1 for 1 min. Temperature significantly affected EPN viability over time. Temperatures around 20 °C preserved optimal conditions, and above 30 °C negatively affected EPN viability, with mortality close to 80% within 90 min at 40 °C. Prodding (measuring survival) yielded higher viability compared to NaCl and no stimulation, which measured activity. Non-lethal stress parameter increased accordingly to stress increment showing potential as EPN stress-marker. CONCLUSION: The study concluded that combined measurement of survival, activity and non-lethal stress should be considered in EPN viability assessments when designing PAE to ensure high efficacy of biocontrol agents. © 2025 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.