Optimization of cryopreservation formulations for the entomopathogenic nematode Steinernema carpocapsae

The short shelf life and storage sensitivity of entomopathogenic nematodes (EPNs) limit their large-scale application as biological control agents and risk the loss of valuable germplasm resources. Cryopreservation offers a potential solution; however, its efficiency is often limited by cryoinjury, resulting in low post-thaw survival rates and reduced infectivity. Current protocols are suboptimal and rarely consider the synergistic effects among cryoprotectants.

We tested three polyols (ethylene glycol, propylene glycol, and glycerol) for the cryoprotection of Steinernema carpocapsae at different concentrations and incubation times. Glycerol exerted the strongest cryoprotective effect, followed by ethylene glycol, while propylene glycol yielded the lowest survival rate. Two-step ethylene glycol pretreatment yielded 15.8 ± 1.5% post-thaw survival, which was higher than trehalose alone but remained far below the best-performing ethylene glycol-trehalose formulation. Trehalose supplementation significantly enhanced survival, particularly in the ethylene glycol-treated group. Orthogonal tests identified two optimal cryopreservation formulations for EPNs: 26% glycerol with 7% trehalose and 108 h of incubation, and 22% ethylene glycol with 2.5% trehalose and 105 min of incubation. Both formulations were also effective in cryopreserving root-knot nematodes, with comparable post-thaw survival of approximately 30%, approximately 1.6-fold higher than that in the untreated control. Virulence assays confirmed that cryopreserved EPNs retained infectivity without significant losses.

Our optimized polyol-trehalose formulation ensured high viability and pathogenicity of cryopreserved EPNs, thereby facilitating germplasm management and improving the practicality of EPN-based biocontrol applications. © 2026 Society of Chemical Industry.