Submitted to: Society for Cryobiology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/15/2005
Publication Date: 12/1/2005
Citation: Rajamohan, A., Yocum, G.D., Leopold, R.A. 2005. Differential gene expression in Mexican fruit flies after cryopreservation [abstract]. Cryobiology. 51(3):406.
Technical Abstract: Potential users of cryopreserved insect germplasm have expressed concern that the vitrification process, including the chemical permeabilization and post storage recovery procedures, may have an effect on overall insect quality. This concern relates to the necessary use of late stage embryos that are cryopreserved when organogenesis is nearing completion. Our previous studies on the post cryopreservation quality of Mexican and Mediterranean fruit flies showed no differences in reproduction, flight ability or genetic diversity but, with the screwworm fly, a first generation low pupal weight and reduced adult emergence was noted . Further, we have observed that of the 5 species of flies that we have cryopreserved thus far, all show significantly slower rates of development to adulthood. Continuing our attempts to resolve apprehension over possible quality issues, we have initiated a study examining differential gene expression in cryopreserved and non-cryopreserved Mexican fruit flies. Profiling differential gene expression has become increasingly popular means to identify the molecular effects of stressors which, in the case of our insect embryo cryopreservation protocol, includes the potential for cold shock, osmotic stress, chemical toxicity and oxygen deprivation. In this study, adult males reared from embryos cryopreserved using the technique of Rajamohan et al.  and untreated controls were used for mRNA isolation. Primary screening for differentially expressed genes was carried out using a modified differential display technique. The majority of the generated gene fragments showed similar expression in both the cryopreserved and untreated flies. Thirteen PCR amplicons of possible differentially regulated genes were isolated and cloned. The BlastX software was used to screen the GeneBank sequence repository for homology. Putative genes displaying down-regulation were identified as ATP synthase, ribosylation factor, ribosomal protein L-4 and L-18, unspecified ras, growth inhibitor and imaginal disc growth factor 4 genes. Conversely, a gene involved in flight muscle development showed up-regulation in the cryopreserved flies. We are affirming these results by Northern blot analysis and are continuing to screen for other differentially expressed genes in cryopreserved insects. We also intend to examine whether differential expression of these and other genes persist in the subsequent generations. It is hoped that this new method of analyzing post cryopreservation quality will alleviate various doubts about the competence of cryopreserved organisms and/or lead to the improvement of the cryopreservation process. (Source of funding: USDA-ARS, Conflict of interest: None declared)  R.A. Leopold, W.B.Wang, D.R. Berkebile, T.P. Freeman. Ann. Entomol. Soc. Amer. 94 (2001) 695.  A. Rajamohan, R.A. Leopold, W.B.Wang, M.Harris, S.D. McCombs, N.C. Peabody, K. Fisher. Cryo-Letters 24 (2003) 125.