**3.5 Vitrification by a glycerol/methanol two-step procedure**

An entirely different treatment methodology was developed primarily for vitrifying the entomopathogenic nematodes in the genera *Steinernema* and *Heterorhabditis*, which are effective as a biological control agent for insect pests in agriculture (Popiel & Vasquez, 1991). *Steinernema carpocapsae* were pre-incubated in 22% glycerol at room temperature for 24 hours and then in ice cold 70% methanol for 10 minutes. After removal of the majority of methanol by centrifugation, concentrated worms in methanol (i.e. 20l) were spread on a small strip of filter paper before plunging into liquid nitrogen for rapid cooling. Up to 95% post-thaw survival rate could be obtained. On the other hand, a post-thaw survival rate of about 55% was obtained on *Heterorhabditis bacteriophora* when the optimal 14% glycerol was used (Popiel & Vasquez, 1991).

Curran et al. (1992) further optimized the protocol by replacing the centrifugation with a filtration step to remove glycerol prior to the methanol incubation. The optimal conditions for glycerol incubation for a number of entomopathogenic nematodes were also determined. Optimal glycerol pre-incubation conditions were determined to be 18% glycerol for 24 hours for *Steinernema carpocapsae*, 17% glycerol for 72 hours for *Heterorhabditis bacteriophora* and 13.8% glycerol for 72 hours for *Steinernema feltiae* and *Steinernema glaseri* (Curran et al., 1992). Other than that, 167 entomopathogenic nematodes were found to be able to survive the cryopreservation treatments, proving the feasibility of cryobanking of these worms. The mean survival rate of the *Steinernema* species is 58% (ranging from 25% to 97%) and that of *Heterorhabditis* species is 51% (ranging from 25% to 87%).

Based on the modification by Curran et al. (1992), Nugent et al. (1996) optimized cryopreservation on seven isolates of *Heterorhabditis*. Up to 8 days of pre-incubation in 11% or 15% glycerol is optimal for cryopreserving a couple of isolates. Nugent (1996) also found that glycerol can be replaced by DMSO in the pre-incubation step. For example, incubation of isolate HI82 in 8% DMSO for 3 days yielded a survival rate of about 80%, similar to those pre-incubated in 15% DMSO.

To the best of our knowledge, unlike mammals, there have been no studies of interactions between nematodes and cryoprotectants. Conversely, different protocols have been developed independently by different groups for specific worm species. Whether the different protocols are indeed applicable to other groups of nematodes or not requires

Cryopreservation of Embryos from Model Animals and Human 273

the outer crystalline chorion layer and the inner vitelline layer (Papassideri et al., 1991). The wax is mainly composed of n-alkanes and methyl-branched alkanes (Nelson & Leopold, 2003), making the eggshell impermeable even to water, thus protecting the embryos from desiccation. Extracting the dechorionated eggs with a wax removing solvent makes the vitelline membrane permeable to water, cytological stains and antibiotics (Schreuders et al., 1996), (Limbourg & Zalokar, 1973). It supports the notion that the wax component in the vitelline layer is the major factor blocking the cryoprotecting molecule from permeating into

To facilitate permeation of cryoprotectants into insect embryos for further cryopreservation protocol development, attempts were made to permeabilize the eggshell but retain the viability of the embryos. Removal of the chorion can be done by exposing the eggs to about 2.5% sodium hypochlorite, without compromising the survival of the embryos (Lynch et al., 1989). Permeabilizing the inner layer of the eggshell with low injury can be done by a 2-step method. Dechorionated eggs of 12-13 hour embryos were rinsed with isopropanol and hexane. The embryos in permeabilized eggshell experienced minimal injury with a survival rate of 75% to 90% in culture medium (Lynch et al., 1989). This procedure resulted in 80%- 95% of the treated eggs being permeabilized to water, ethylene glycol, propylene glycol, glycerol and DMSO. Mazur et al. (1992b) further optimized the two-step method to allow permeation of common cryoprotectants such as ethylene glycol and glycerol. The best result was obtained by exposing the dechorionated 12-14 hour embryos to 0.3%-0.4% 1-butanol in n-heptane for 90 seconds. At least 90% permeabilization and 80% survival can be obtained. Older embryos between 14-16 hours are much less sensitive to the above procedures, which are lethal to 3 hour embryos. Using procedures similar to Mazur (1992b), with decreased concentration of sodium hypochlorite in dechorionation and replacing isopropanol with air drying, all mosquito (*Anopheles gambiae*) embryos at 15-19 hours can be permeabilized in ethylene glycol with an acceptable survival rate of 30% (Valencia et al., 1996). On the other hand, heptane treatment seemed to be detrimental to the greater wax moth (*Galleria mellonella*) embryos. Replacing the heptane treatment with incubation in 1.25% sodium hypochlorite with 0.08% Tween-80 for 2 minutes permeabilized the moth embryos with 68% survival (Cosi et al., 2010). Determination of the optimal permeabilization procedures and the optimal embryonic stage to be permeabilized in different insecta species has opened the

door for efficient cryopreservation of this largest class of animals on land.

*Drosophila* embryos are highly sensitive to chilling. When 15 hour eggs were incubated at - 15°C, 50% of the embryos died within an hour even in the absence of ice formation. When younger embryos at 3 hours and 6 hours were cooled to the same temperature, the chilling injury increased dramatically (Mazur et al., 1992c). All 12 hour embryos died at -25°C when cooled at 1°C per minute. A similar phenomenon was found in case of honey bee (*Apis mellifera*) embryos (Collins & Mazur, 2006). The slow cooling approach that requires time for efflux of intracellular water osmotically to avoid intracellular ice formation is, therefore, theoretically impractical for handling insect embryo cryoprotection. It was estimated that cooling the embryos faster than 300°C per second can circumvent the chilling injury by shortening the time the embryo stays at such a low temperature. However, lethal

**4.3 Chilling sensitivity of insect embryos** 

the embryos.

further investigation. Nonetheless, we can interpret from the protocols that nematodes are generally resistant to cryoprotectant toxicity because most protocols involve a relatively long incubation time in cryoprotecting solutions. Surprisingly, most of the protocols do not involve removal of the cuticle, which is well known for its poor permeability, though successful cryoprotection requires the presence of enough intracellular concentration of cryoprotectant. It is possible that the cryoprotectants themselves can permeabilize the cuticle and then permeate into the cells beneath. Or the cryoprotectant enters the worm via the oral opening and the gut, into the rest of the body. Equally possible is that the "cryoprotectants" did not act as a cryoprotectant *per se* but acted as inducers to trigger the production of natural cryoprotectant in the worm. Therefore, thorough permeation of the chemicals was not required.
