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Routine Cryopreservation of Isolates of Steinernema and Heterorhabditis spp
Abstract:
Infective-stage juveniles of Steinernema and Heterorhabditis spp. were cryopreserved using two-stage incubation in glycerol and 70% methanol before storage in cryotubes in liquid nitrogen. Optimal glycerol concentrations and incubation times for survival were determined for different species, but acceptable survival of all species and isolates of entomopathogenic nematodes can be obtained using 15% (w/w) glycerol and incubation for 48 hours. Mean survival was 69% for isolates of Steinernema and 68% for isolates of Heterorhabditis (n = 84). The maximum survival recorded was 97% for S. feltiae K254 stored in liquid nitrogen for 12 months.
Insights
Cryopreservation of entomopathogenic nematodes using glycerol and methanol in liquid nitrogen offers high survival rates. This method ensures the long-term viability of Steinernema and Heterorhabditis species for biological control applications.
Area of Science:
- Nematology
- Cryobiology
- Biological Control
Background:
- Entomopathogenic nematodes (EPNs) are vital biological control agents.
- Effective long-term storage methods are crucial for EPNs like Steinernema and Heterorhabditis spp.
- Cryopreservation in liquid nitrogen is a promising storage technique.
Purpose of the Study:
- To optimize cryopreservation protocols for infective-stage juveniles of Steinernema and Heterorhabditis spp.
- To determine optimal glycerol concentrations and incubation times for nematode survival.
- To establish a reliable method for long-term storage of EPNs.
Main Methods:
- Two-stage incubation of EPNs in glycerol and 70% methanol.
- Storage of cryopreserved nematodes in cryotubes in liquid nitrogen.
- Evaluation of survival rates across different species and isolates.
Main Results:
- Acceptable survival for all tested EPN species and isolates was achieved with 15% (w/w) glycerol and 48-hour incubation.
- Mean survival rates were 69% for Steinernema and 68% for Heterorhabditis isolates (n=84).
- Maximum survival reached 97% for Steinernema feltiae K254 after 12 months of storage.
Conclusions:
- A two-stage cryopreservation method using glycerol and methanol is effective for Steinernema and Heterorhabditis spp.
- The established protocol ensures high survival rates and long-term viability of EPNs.
- This method supports the sustained availability of EPNs for integrated pest management programs.
