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

BACKGROUNDConventional 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.RESULTSThree 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.CONCLUSIONThe 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.