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  • Cited by 5
  • Print publication year: 2010
  • Online publication date: May 2010

13 - A template for insect cryopreservation




While there is a growing number of insect species that have been successfully cryopreserved during the almost two decades that have elapsed since publication of Insects at Low Temperature, practical use of the methodology has generally not been forthcoming. The benefits of having the capability to place insects into storage indefinitely are considerable, ranging from reducing rearing costs to preserving valuable genetic resources. However, traditional continuous rearing practices still prevail as the primary means to maintain multiple colonies of insects, even though cryopreservation protocols for some species are now available. It is understandable that scientists and insectary managers would be reluctant to make significant changes in rearing methods without adequate assurance that irreplaceable research stocks or production lines were not lost. With this in mind, we wish to use this communication to demonstrate how a cryopreservation protocol is developed and utilized for practical applications. Thus, we hope to help alleviate the concerns about the use of existing protocols and stimulate additional research on developing cryogenic technology for the long-term storage of insects important to agriculture and the scientific community.

The insect used as the example for this case study is the New World Screwworm, Cochliomyia hominivorax, an ectoparasitic calliphorid that causes myiasis in man and animals (Bishopp, 1915). It is the first insect to be eliminated through an area-wide release of mass-produced sterile insects and it no longer exists in North and Central America (Wyss, 2000).

Allen, M. L., Handler, A. M., Berkebile, D. R. and Skoda, S. R. (2004). piggyBac transformation of the New World screwworm Cochliomyia hominivorax, produces multiple distinct mutant strains. Journal of Medical and VeterinaryEntomology 18, 1–9.
Berkebile, D. R., Chirico, R. J. and Leopold, R. A. (2000). Permeabilization of Cochliomyia hominivorax (Diptera: Calliphoridae) embryos. Journal of Medical Entomology 37, 968–972.
Bishopp, F. C. (1915). Flies which cause myiasis in man and animals – some aspects of the problem. Journal of Economic Entomology 8, 317–329.
Galvin, T. J. and Wyss, J. H. (1996). Screwworm eradication program in Central America. Annals of the New York Academy of Sciences 791, 233–240.
Heacox, A. E., Leopold, R. A. and Brammer, J. D. (1985). Survival of house fly embryos cooled in the presence of dimethyl sulfoxide. CryoLetters 6, 305–312.
Jutte, N. H., Heyse, P., Jansen, H. G., Bruining, G. J. and Zeilmaker, G. H. (1987). Vitrification of human islets of Langerhans. Cryobiology 24, 403–411.
Isachenko, V. V., Isachenko, E. F., Ostashko, F. I. and Grishchenko, V. I. (1997). Ultrarapid freezing of rat embryos with rapid dilution of permeable cryoprotectants. Cryobiology 34, 157–164.
Isachenko, V., Montage, M., Isachenko, E., Nawroth, F., Dessole, S. and Ven, H. (2004). Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration. Human Reproduction 19, 660–665.
Leopold, R. A. (1991). Cryopreservation of insect germplasm: cells, tissues and organisms. In Insects at Low Temperature, ed. Lee, R. E. and Denlinger, D. L.. New York, NY: Chapman and Hall, pp. 379–407.
Leopold, R. A. (2007). Cryopreservation of nonmammalian metazoan systems. In Advances in Biopreservation, ed. Baust, J. G. and Baust, J. M.. Boca Raton, FL: CRC Taylor and Francis Group, pp. 271–298.
Leopold, R. A., Rajamohan, A., Shelly, T. E. and Handler, A. M. (2010). Quality testing of three species of tephritid fruit flies after embryo cryopreservation. Annals of Entomological Society of America (in press).
Leopold, R. A., Wang, W. B., Berkebile, D. R. and Freeman, T. P. (2001) Cryopreservation of embryos of the New World Screwworm, Cochliomyia hominivorax (Diptera: Calliphoridae). Annals of Entomological Society of America 94, 695–701.
Mazur, P. (1990). Equilibrium, quasi-equilibrium and nonequilibrium freezing of mammalian embryos. Cell Biophysics 17, 53–92.
Mazur, P. (2004). Principles of cryobiology. In Life in the Frozen State, ed. Fuller, B. J., Lane, N. and Benson, E. E.. Boca Raton, FL: CRC Taylor Francis Group, pp. 3–65.
Mazur, P., Cole, K. W., Hall, J. W., Schreuders, P. D. and Mahowald, A. P. (1992). Cryobiological preservation of Drosophila embryos. Science 258, 1932–1935.
Mazur, P., Cole, K. W., Schreuders, P. D. and Mahowald, A. P. (1993) Contributions of cooling and warming rate and developmental stage to the survival of Drosophila embryos cooled to −205 °C. Cryobiology 30, 45–73.
Meryman, H. T. (1971). Cryoprotective agents. Cryobiology 8, 173–183.
Meryman, H. T. (2007). Cryopreservation of living cells: principles and practice. Transfusion 47, 935–945.
Miles, J. E. and Bale, J. S. (1995). Analysis of chilling injury in Aphidoletes aphidimyza. Cryobiology 32, 45–73.
Moon, I., Fujikawa, S. and Horie, Y. (1996). Cryopreservation of Chymomyza costata larvae (Diptera: Drosophilidae) at −196 °C with extracellular freezing. CryoLetters 17, 105–110.
Nelson, D. R. and Leopold, R. A. (2003). Composition of the surface hydrocarbons from the vitelline membrane of dipteran embryos. Comparative Biochemistry and Physiology 136, 210–308.
Nishizawa, S., Sakai, A.Amano, Y. and Matsuzawa, T. (1993). Cryopreservation of asparagus (Asparagus officinalis L.): embryogenic suspension, cells and subsequent plant regeneration by vitrification. Plant Science 91, 67–73.
Nunamaker, R. A. and Lockwood, J. A. (2001). Cryopreservation of embryos of Culicoides sonorensis (Diptera: Ceratopogonidae). Journal of Medical Entomology 38, 55–58.
Ogawa, Y.Suzuki, H., Sakurai, N.Aoki, K., Saito, K.and Shibata, D. (2008) Cryopreservation and metabolic profiling analysis of Arabidopsis T87 suspension-cultured cells. CryoLetters 29, 427–436.
Rajamohan, A. and Leopold, R. A. (2007). Cryopreservation of Mexican fruit flies by vitrification: stage selection and avoidance of thermal stress. Cryobiology 54, 44–54.
Rajamohan, A., Leopold, R. A., Wang, W. B., Harris, M., McCombs, S. D., Peabody, N. C. and Fisher, K. (2003). Cryopreservation of Mediterranean fruit fly embryos. CryoLetters 24, 125–132.
Rajamohan, A., Yocum, G. D. and Leopold, R. A. (2005). Differential gene expression in Mexican fruit flies after cryopreservation. Cryobiology 51, 406.
Rall, W. F. (1987). Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 24, 387–402.
Rall, W. F. and Fahy, G. M. (1985) Ice-free cryopreservation of mouse embryos at −196 °C by vitrification. Nature 313, 573–575.
Schneider, I. (1964). Differentiation of larval Drosophila eye-antennal discs in vitro. Journal of ExperimentalZoology 156, 91–104.
Sømme, L. (1964). Effects of glycerol on cold hardiness in insects. Canadian Journal of Zoology 42, 87–101.
Steponkus, P. L. and Caldwell, S. (1993). An optimized procedure for the cryopreservation of Drosophila melanogaster embryos. CryoLetters 14, 377–380.
Suszkiw, J. (2005) Frozen flies safeguard research, screwworm eradication efforts. Agricultural Research 53, No. 2, 14–15.
Takahashi, T., Hirsh, A., Erbe, E. F., Bross, J. B., Steere, R. L. and Williams, R. J. (1986). Vitrification of human monocytes. Cryobiology 23, 103–115.
Wagtendonk-De Leeuw, A. M., Daas, J. H. G., Kruip, Th. A. M. and Rall, W. F. (1997). Comparison of the efficacy of conventional slow freezing and rapid cryopreservation methods for bovine embryos. Cryobiology 32, 157–167.
Wang, W. B., Leopold, R. A., Nelson, D. R. and Freeman, T. P. (2000). Cryopreservation of Musca domestica (Diptera: Muscidae) embryos. Cryobiology 41, 153–166.
Wessel, M. T. and Ball, B. A. (2004). Step-wise dilution for removal of glycerol from fresh and cryopreserved equine spermatozoa. Animal Reproduction Science 84, 147–156.
Wusteman, M. C., Simmonds, J., Vaughan, D. and Pegg, D. E. (2008). Vitrification of rabbit tissues with propylene glycol and trehalose. Cryobiology 56, 62–71.
Wyss, J. H. (2000). Screwworm eradication in the Americas. Annals of the New York Academy of Sciences 916, 186–193.
Yildiz, C., Ottaviani, P., Law, N., Ayearst, R., Liu, L. and McKerlie, C. (2007). Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse. Reproduction 133, 585–595.