Submitted to: Cryobiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/3/2006
Publication Date: 2/26/2007
Citation: Rajamohan, A., Leopold, R.A. 2007. Cryopreservation of Mexican fruit flies by vitrification: Stage selection and avoidance of thermal stress. Cryobiology. 54(1):44-54. Interpretive Summary: This study reports the development of a procedure for long-term storage of Mexican fruit fly embryos in liquid nitrogen. The most important feature of the procedure that dictates success or failure is to identify the correct stage of embryonic development that is amenable for treatment. To correctly identify the exact stage for treatment, we searched for visual markers in or on the embryos that would make it easier to determine when to implement the procedure. These markers were found to be related to disappearance of yolk in the midgut and formation of mouth hooks on the head of the embryo. Hatching after cryopreservation was nearly 80% when embryos were hand selected from a population of all the same age for the correct stage for treatment by using our markers. Other aspects of the study included development of information on the cryoprotectants, cooling and recovery of the embryos. This information explains why the particular strategies used in this procedure are important to gaining successful embryo cryopreservation of this insect and the other insect species that have been previously cryopreserved with similar protocols developed in our laboratory. Recovery of Mexican fruit fly embryos after more than 1 year of storage in liquid nitrogen showed no difference in hatching when compared to recently cryopreserved embryos.
Technical Abstract: This report presents details of a vitrification methodology for the cryopreservation of Mexican fruit fly, Anastrepha ludens. The overall summary of the data indicates that selecting the correct stage for cryopreservation is the most important criterion. The most crucial aspect in the selection of the correct stage was balancing depletion of the gut yolk content before the embryonic cuticle developed. Embryogenesis was divided into 4 stages aged between 90 and 120 hours after incubation at 21.7 deg. C. The classification was based on the intestinal yolk content and development of mandibulo-maxillary complex. Stages having low mid-gut yolk content and the appearance of mouth hooks were found to be the most suitable for cryopreservation. This was true only of samples incubated at 21.7 and 24 deg. C. Embryos developing at 30 deg. C had asynchronous cuticle formation relative to gut development which resulted in significantly lower hatching after cryopreservation. Vitrification of embryos by direct quenching in liquid nitrogen was less effective than quenching after annealing the samples in liquid nitrogen vapor. Quenched samples of vitrification solutions exhibited fractures that occurred less frequently when the solutions were annealed and which also contained polyethylene glycol. Hatching of vitrified embryos stored in liquid nitrogen for over 12 months was not statistically different from those held for only a few minutes. Our protocol yielded normalized hatching rates as high as 61%. Selecting the exact stage for cryopreservation using our markers from a population of embryos of the same age gained by collection from a large colony resulted in nearly 80% of the embryos hatching into larvae.