**4.1 Development of insect embryos**

*Insecta* is a class with the greatest interest among the *Arthropoda*. Pathogenic vectors associating mosquitoes, flies and bugs are of interest in medical and healthcare related research. Honeybee, silk moth, beetle and other moths are of economic interest to the beekeeping industry, silk industry and pest control in agriculture, respectively. The fruit fly *Drosophila melanogaster*, a species of the order *Diptera*, is chosen as an insect model in genetic studies because of its ease of keeping, handling and observing. It has a short life cycle and is fertile through out the year. A complete life cycle involves four distinct stages of development, which takes 8.5 days for a *Drosophila* egg to reach adulthood at 25C.

A *Drosophila melanogaster* egg is about 0.18mm in width and 0.5mm in length, occupying a volume of about 9nl. The laid egg is a single cell with about 6,000 nuclei which later migrate to the plasma membrane to form a syncytial blastoderm at around 2 hours. Cell membranes form between the nuclei at around 2.5 hours. As gastrulation occurs, a ventral furrow and a cephalic furrow form. The midgut invaginates followed by a germ band extension (3-7 hours), stomodeal invagination (5-7 hours), germ band shortening, foregut and hindgut deeply invaginate (9-10 hours). The ectoderm closes dorsally and the head involutes at about 10-13 hours (Figure 3B). At around 13 hours, organogenesis has already begun in the 50,000-cell embryos. Organogenesis is completed and the gut regions are joined between 15 and 22 hours (i.e. at hatching) (Markow et al., 2009; Weigmann et al., 2003; Grumbling & Strelets, 2006). The hatched larva experiences three instar larval phases, before the skin of the third-instar larva hardens and encapsulates the animal to form a puparium. It takes four days for the third larva to transform into an adult by metamorphosis. At the end of metamorphosis, the adult of about 2.5mm length emerges from the puparium.

#### **4.2 Permeabilization of insect embryos to cryoprotectants**

The outermost layer of the eggshell of an insect embryo is a porous proteinacious chorion with a thickness ranging from 300-500nm in *Drosophila* to more than 40m in saturniid moth (Magaritis et al., 1980; Fehrenbach, 1995). Underneath are: (1) the innermost crystalline chorion layer of about 40nm in *Drosophila* (Papassideri & Margaritis, 1996), (2) the waterproofing wax layer (Beament, 1946; Slifer, 1948) and (3) the vitelline layer (Papassideri et al., 1991). The wax layer is only 5nm in thickness in *Drosophila* and is intercalating with

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

*Insecta* is a class with the greatest interest among the *Arthropoda*. Pathogenic vectors associating mosquitoes, flies and bugs are of interest in medical and healthcare related research. Honeybee, silk moth, beetle and other moths are of economic interest to the beekeeping industry, silk industry and pest control in agriculture, respectively. The fruit fly *Drosophila melanogaster*, a species of the order *Diptera*, is chosen as an insect model in genetic studies because of its ease of keeping, handling and observing. It has a short life cycle and is fertile through out the year. A complete life cycle involves four distinct stages of

A *Drosophila melanogaster* egg is about 0.18mm in width and 0.5mm in length, occupying a volume of about 9nl. The laid egg is a single cell with about 6,000 nuclei which later migrate to the plasma membrane to form a syncytial blastoderm at around 2 hours. Cell membranes form between the nuclei at around 2.5 hours. As gastrulation occurs, a ventral furrow and a cephalic furrow form. The midgut invaginates followed by a germ band extension (3-7 hours), stomodeal invagination (5-7 hours), germ band shortening, foregut and hindgut deeply invaginate (9-10 hours). The ectoderm closes dorsally and the head involutes at about 10-13 hours (Figure 3B). At around 13 hours, organogenesis has already begun in the 50,000-cell embryos. Organogenesis is completed and the gut regions are joined between 15 and 22 hours (i.e. at hatching) (Markow et al., 2009; Weigmann et al., 2003; Grumbling & Strelets, 2006). The hatched larva experiences three instar larval phases, before the skin of the third-instar larva hardens and encapsulates the animal to form a puparium. It takes four days for the third larva to transform into an adult by metamorphosis. At the end of

development, which takes 8.5 days for a *Drosophila* egg to reach adulthood at 25C.

metamorphosis, the adult of about 2.5mm length emerges from the puparium.

The outermost layer of the eggshell of an insect embryo is a porous proteinacious chorion with a thickness ranging from 300-500nm in *Drosophila* to more than 40m in saturniid moth (Magaritis et al., 1980; Fehrenbach, 1995). Underneath are: (1) the innermost crystalline chorion layer of about 40nm in *Drosophila* (Papassideri & Margaritis, 1996), (2) the waterproofing wax layer (Beament, 1946; Slifer, 1948) and (3) the vitelline layer (Papassideri et al., 1991). The wax layer is only 5nm in thickness in *Drosophila* and is intercalating with

**4.2 Permeabilization of insect embryos to cryoprotectants** 

not required.

**4. Cryopreservation of insect embryos** 

**4.1 Development of insect embryos** 

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 the embryos.

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.

#### **4.3 Chilling sensitivity of insect embryos**

*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

Cryopreservation of Embryos from Model Animals and Human 275

The zygote of zebrafish (*Brachydanio rerio*) is about 0.7 mm in diameter when fertilization occurs. A few minutes later, the chorion swells to increase the diameter to about 1.2 mm, without much alteration in thickness, generating a significant vitelline space. The cytoplasm segregates to form the animal pole and the vegetal yolk with an approximate total volume of 128nl, not including the vitelline space and the chorion (Leung et al., 1998). The first cell cleavage occurs in the animal pole at about 45 minutes after fertilization. The blastomere gets divided five more times synchronously, each at about 15 minutes interval, producing a blastoderm with 64 cells in 2 hours. The daughter cells increase in number with a decrease in cell size. The blastomeres arrange themselves in a single cell layer before the fifth cleavage. Afterwards, newly formed daughter cells overlap with each other in the blastoderm. The multi-cell layered blastoderm spread over the yolk, reaching 30% epiboly at 4.7 hours and 50% epiboly at 5.25 hours (Figure 3C). At the gastrula period, epiboly continues at 5.3 hours. Two germ layers, i.e. epiblast and hypoblast, are formed by morphogenetic movement of involution, convergence and extension. Epiboly reaches 90% at 9 hours. At the end of this period, the tail bud and neural plate starts to form. The volume of the epiboly remains constant from 40% epiboly to 100% epiboly. Entering the segmentation period at 10 hours, segmentation processes such as formation of neuromeres, somites and the pharyngeal arch primordia occur. The embryo volume increases to 0.23 mm3 at the sixsomite stage at 12 hours when organogenesis starts (Hagedorn et al., 1997c). At the end of the 14 hour-period, the yolk largely reduces and tail movement can be seen. Pigment can be identified after 36 hours. At the third day, the primary organogenesis completes. Cartilage in the head and pectoral fin develops while hatching occurs anytime in the third day

Fish embryos are composed of several components with distinct physical properties. They include the highly dynamic cellular part on the animal pole which contributes entirely to the future animal body, the yolk surrounded by the yolk syncytial layer occupying the majority of the early stage embryo and the chorion as the outermost mechanical protective shield. From 40-100% epiboly, the blastoderm and the yolk occupy 18% and 82% of the dechorionated embryo, respectively, in zebrafish (*Brachydanio rerio*). At the six-somite stage, the volume of the blastoderm increases to about 40%, leaving the yolk occupying 60% of the embryo (Hagedorn et al., 1997c). Between the chorion and the embryo is the perivitelline space filled with liquid with a chemical composition virtually identical to the surrounding medium (Rawson et al., 2000). The complexity of the teleost embryos is further increased by the unbalanced partial density of water in different compartments. At the six-somite stage, the blastoderm occupies about 40% of the dechorionated embryos but water constitutes 82% of its volume. Conversely, the yolk occupies about 60% of the volume of the dechorionated embryos but only 42% of this is constituted by water (Hagedorn et al., 1997b). It was estimated that the osmotically inactive volume in the onesomite stage and six-somite stage embryos are 72.9% and 82.6%, respectively (Zhang &

**5. Cryopreservation of teleost embryos** 

**5.1 Development of teleost embryos** 

(Kimmel et al., 1995).

Rawson, 1998).

**5.2 Complexity of teleost embryos** 

intracellular ice forms at such a cooling rate when using the standard concentration of cryoprotectants (Mazur et al., 1992c). Vitrification is, therefore, the only possible way to cryopreserve insect embryos.
