Submitted to: Journal of Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/6/2022
Publication Date: N/A
Interpretive Summary: Numerous insects are economically important due to their role in pollination, disease transmission and as model organisms to understand biological processes. Research involving the common fruit fly, Drosophila, has led to significant advancements in human health and crop protection. In turn, research labs and stock centers have accumulated over 100,000 different valuable strains of Drosophila. However, maintaining large numbers of Drosophila colonies is labor intensive and costly, such that alternative storage methods are needed. One possibility is to use cryopreservation, or low temperature storage, of fruit fly embryos. The procedure to cryopreserve fruit fly embryos was developed nearly 30 years ago, but it is unknown if it is applicable to all strains of Drosophila. Successful cryopreservation requires precise timing of embryonic development because the timeframe in which embryos are cryopreservable persists for only a few minutes. In this study, we set about to determine a method to precisely gather as many embryos as possible in the right developmental stage in two different strains of Drosophila. We then measured the hatch percentage of cryopreserved embryos and the percentage emergence of flying adults from the larvae obtained from frozen embryos. Our studies identified a new cryopreservation technique that lowers labor costs and yields significantly higher numbers of flies than the conventional method.
Technical Abstract: Embryonic selection for vitrification and cryostorage in Drosophila and other dipterans is generally carried out by gross observation of the embryonic development at a constant temperature. In this study, the effect of embryo developmental temperature (19, 20 and 21°C) on the stage specific convergence of the embryonic development to the developmental stages 15-17, that is useful for cryopreservation, was studied in a flightless mutant strain of Drosophila melanogaster and compared with the wild Ore-R strain. The temperature that allowed for the best convergence to stage 16 was chosen for further selection and treatment of the embryos. The converged embryos (SS) were directly treated or further manually sorted (MS) for the requisite developmental stage to reduce the number of non-convergent embryos. These selected embryos were then permeabilized and cryopreserved. While at all the three incubation temperatures the embryos exhibited convergence peaks, it was only at 20°C and at hour 22 that a maximum number of stage 16 embryos converged and remained at a much higher proportion than the other developmental stages in both the strains. When permeabilized, MS embryos showed higher mean viability and hatching proportion compared to SS embryos (wingless: ~70 vs. ~0.58; Ore-R: ~0.77 versus ~0.55). Upon vitrification, the manually selected embryos hatched and survived at significantly higher mean rates than the converged embryos at stage 16 (wingless: ~0.46 vs. ~0.14; Ore-R: ~0.61 vs. <0.27) after adjusting for permeabilization mortality. The maximum proportion hatch after vitrified storage that could be obtained by this method was 0.74 for both the wingless and Ore-R strains. More than 55% of the larvae pupated and >72% of the pupae eclosed in MS and vitrified wingless stage 16. In Ore-R, well over 85% of the larvae pupariated and eclosed as flight capable flies.