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United States Department of Agriculture

Agricultural Research Service

Title: Cryopreservation of Dipteran Insects: Development and Evaluation

Author
item Leopold, Roger

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 17, 2004
Publication Date: October 20, 2004
Citation: Leopold, R.A. 2004. Cryopreservation of dipteran insects: development and evaluation. Meeting Abstract. October 18-23, 2004. St. Petersburg, Russia. In: Materials of the International Conference "Preservation of Genetic Resources" 46(9):760-761.

Technical Abstract: On a world-wide basis, many of the most important insect pests affecting humans, domestic animals and crops are dipteran species. Mosquitoes, screwworms, midges and the tephritid, filth, and tsetse flies are but a few examples of these destructive and, in some cases, life-threatening pests. Some of the dipteran pests are either being eradicated or controlled by the sterile insect technique (SIT). The SIT involves mass releases of sexually sterile insects that have been reared under factory-like conditions and then dispersed at a high ratio of sterile insects to the comparable wild species to gain control through infertile matings. Developing cold storage protocols to support the rearing regimes for factory-propagated insects can be useful or even a required strategy to ensure that production is economical, reliable, and yields quality insects. Further, using cryopreservation to preserve insect germplasm for purposes of economizing the rearing and maintenance of laboratory research colonies and for the conservation of genetic resources for future generations is also an important endeavor. To date, 7 species of dipterans representing 4 families have been successfully cryopreserved in the embryonic stage and, of these 7 species, protocols for Musca domestica, Cochliomyia hominivorax, Ceratitis capitata, Anastrepha suspensa, and A. ludens have been developed in our laboratory (Wang et al., 2000; Wang et al., 2001; Leopold et al., 2001; Rajamohan et al., 2003). Because of the inherent barriers to using conventional cryopreservation techniques that these dipteran embryos exhibit (Leopold, 1991), development of a protocol for liquid nitrogen storage requires that each protocol be adapted or modified to accommodate the characteristics of each species. Some of the major barriers to using cryopreservation technology include: membrane impermeability, chilling sensitivity, abundance of yolk, and post storage fragility. The techniques that are used to deal with membrane impermeability involve chorion removal and membrane lipid extraction. For overcoming the chilling sensitivity of embryos equipped with abundant yolk, correct stage selection and the use of vitrification technology is required. And, to combat post storage recovery weakness, providing an adequate thawing medium and a fortified larval diet is necessary for obtaining sufficient numbers of adults to recolonize a particular species. The yield of adults after cryopreservation during their embryonic stage varies between species and strains within species. Recent work with 15 different screwworm strains, including wild-type, mutant, and transgenic forms, showed that hatching of cryopreserved embryos can vary between 20-78%. Our evaluation of the quality of the cryopreserved insects and their progeny was done under laboratory and field conditions. These tests included assessments of pupal weight, sex ratio, survival, fertility, fecundity, mating propensity and competitiveness, flight ability and endurance, and genetic diversity. Of all these observations, we found that cryopreserved screwworms had a 15% lower pupal weight which returned to the control level during the next generation. Also, there was an indication that cryopreservation of the Mediterranean fruit fly, C. capitata, TSL strain can produce an altered sex ratio which probably related to the incubation temperature of the embryos prior to treatment. With all other tests, we found no statistical differences between the cryopreserved insects or their progeny when compared to the control groups. In summary, we have tested 5 species of flies cryopreserved as embryos or as progeny of cryopreserved parents for a number of indicators of fitness or quality. Based on the results, there is little to suggest that permanent damage or change occurs when using an insect cryopreservation protocol such has been developed in our laboratory. References: Leopold, R.A. (1991) Cryopreservation of Insect Germplasm: Cells, Tissues and Organisms. In: Insects at Low Temperature. (Eds. R.E. Lee and D.L. Denlinger) Chapman & Hall, pp. 379-407. Leopold, R.A., Wang, W.B., Berkebile, D.R., Freeman, T.P. (2001) Cryopreservation of embryos of the New World Screwworm Cochliomyia hominivorax (Diptera: Callophoridae). Ann. Entomol. Soc. Amer. 94: 695-70. Rajamohan, A., Leopold, R.A., Wang, W.B., Harris, M., McCombs, S.D., Peabody, N.C., Fisher, K. (2003) Cryopreservation of Mediterranean fruit fly embryos. Cryo-Letters. 24: 125-132. Wang, W.B., Leopold, R.A., Nelson, D.R., Freeman, T.P. (2000) Cryopreservation of Musca domestica (Diptera: Muscidae) embryos. Cryobiology. 41: 153-166. Wang, W.B., Leopold, R.A., Handler, A.M., McCombs, S.D., Rajamohan, A., Freeman, T.P. (2001) Cryopreservation of fruit fly (Tephritidae) embryos. Cryobiology. 43:339-340.

Last Modified: 9/10/2014
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