**3.1 Development of the nematode**

268 Current Frontiers in Cryobiology

Fig. 4. Morphological changes of mammalian (mouse) preimplantation embryos in response to cryoprotectant treatments (for vitrification) and rehydration. Panel A-E: zygote (1-cell); panel F-J, pre-compact morula (8-cell); panel K-O, compact morula (8-cell); panel P-T, expanded blastocyst. Individual embryos were held by a glass micropipette by a slight suction under physiological isotonic medium (A, F, K and P). Vitrification solutions containing low concentration of permeating cryoprotectants and high concentration of permeating

cryoprotectants plus non-permeating cryoprotectants were applied to the surroundings of the embryos, sequentially. About 30 seconds after the application of solution containing low concentration of permeating cryoprotectants, the embryos osmotically shrunk to minimal volumes (panel B, G, L and Q). The embryos (except the blastocyst) later re-expanded to a size closer to the original volumes, after an additional 30 seconds, when cryoprotectants and water re-entered the cells passively (panel C, H, M and R). After addition of the final vitrification solution containing high concentration of permeating cryoprotectants and non-permeating cryoprotectants, the embryos were further dehydrated by osmosis and shrunk without re-

expansion. (panel D, I, N and S) High concentration of intracellular permeating

gained normal morphologies (E, J, O and T).

cryoprotectants was achieved. Rapid cooling is normally done at this stage to vitrify the embryos but such cooling was not done in this demonstration. Step-wise rehydration of the embryos was done after dehydration to imitate the recovery steps after thawing the embryos from vitrification. After rehydration, the embryos re-expanded to their original size and reNematoda belong to the ecdysozoa, sharing the same clade with arthropoda. The members range from free living species to parasitic species in plants and animals. The life cycle of the nematode is generally divided into five morphological stages. Each successive larval stage is preceded by a molting process to remove the collagenous cuticle from the former larval stage. At hatching, the first-stage larva (Figure 3 A) consisting of 558 cells is under the protection by a cuticular layer. The animal grows in size after each hatching. In adulthood, a reproductive hermaphrodite is about 1mm long, 0.06mm in diameter, containing about 1,000 somatic cells. In comparison, a first-stage larva is about 0.37mm in length and 0.025mm in diameter.

Under favorable conditions, the development of the animal continues through the first- to the fourth-larval stage and finally to the reproductive adults. In many parasitic species such as the entomopathogenic species, the third-stage larvae are juveniles that are infective to their hosts. Under unfavorable conditions, i.e. outside the host body, a second-stage larva develops into the third-stage infective juvenile but retains the cuticle from the last larval stage to form a sheath. The entire animal is enclosed in the sheath until a suitable host is infected.
