The effect of cryopreservation on the lethal mutation rate in Drosophila melanogaster

Abstract

Although cryopreservation is routinely used for the storage of a range of biological organisms, few studies have been conducted to determine whether cryopreservation increases the frequency of mutation. A procedure for the cryopreservation of Drosophila melanogaster embryos has recently been developed. Cryopreservation of D. melanogaster is of special interest to geneticists and evolutionary biologists because it would make it possible to assay control and experimental populations simultaneously during long-term studies. Before cryopreserved embryos can be used for such studies, it is first necessary to show that cryopreservation is not mutagenic. We tested for mutagenic effects or cryopreservation in D. melanogaster embryos with an X-linked, recessive lethal assay. The mutation rates of cryopreserved and control flies were not significantly different. We can be 95% certain that cryopreservation does not increase mutation by a factor greater than 2.39. This is the first quantitative estimate of the mutagenic effect of cryopreservation on the germ line of a metazoan. The results are reassuring when considering the genetic impact of cryopreservation on mammalian gametes and embryos.

Mutation accumulation and the effect of copia insertions in Drosophila melanogaster

“…In previous work, rates of success in reviving embryos of a particular test genotype were as high as 45 % (Steponkus & Caldwell, 1993). Our results (here and Houle et al, 1997) are the only attempts we know of to revive embryos from cryopreservation after more than a few days. Additional attempts to use the method are needed before it is discarded.…”

“…Mutation during cryopreservation is unlikely to have been important. We estimated that the lethal mutation rate during the cryopreserved generation was equal to that in a normal generation (with an upper 95 % limit of an increase by a factor of two ; Houle et al, 1997), making mutation an unlikely cause of fitness decline. Viability selection during cryopreservation should not affect fitness because our controls were inbred lines that would have very little genetic variation.…”

“…IV+ has been maintained in ten half-pint bottles since 1975 and was rendered homosequential in 1978. In 1992, a spontaneous ebony mutation (Lindsley & Zimm, 1992) was found at appreciable frequency in one replicate of IV+, and homozygotes for this recessive allele were backcrossed to IV+ flies in four rounds to form a homozygous ebony population on an outbred IV+ background (Houle et al, 1997). The IV+ population has a cytotype of R and weak P .…”

“…Shortly before the establishment of the MA lines, samples of each line were cryopreserved as embryos (Houle et al, 1997). Each line therefore had to be expanded to approximately 10 000 flies to provide many eggs at the proper age.…”

“…We accumulated mutations by brother-sister matings of flies of two inbred lines, similar to the approach taken by Chavarrı´as et al (2001). First, we used a cryopreserved sample (Houle et al, 1997) of the original population as a control, which was revived before each fitness assay. Second, we used de novo insertions of the transposable element (TE) copia to detect directly a substantial proportion of the spontaneous deleterious mutations occurring in one of our lines.…”