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United States Department of Agriculture

Agricultural Research Service

Research Project: INSECT CRYOPRESERVATION, DORMANCY, GENETICS AND BIOCHEMISTRY

Location: Insect Genetics and Biochemistry Research

Title: Cryopreservation of embryos of Lucilia sericata (Diptera: Calliphoridae)

Authors
item Rajamohan, Arun -
item Rinehart, Joseph
item Leopold, Roger

Submitted to: Journal of Medical Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 7, 2014
Publication Date: March 1, 2014
Repository URL: http://handle.nal.usda.gov/10113/58531
Citation: Rajamohan, A., Rinehart, J.P., Leopold, R.A. 2014. Cryopreservation of embryos of Lucilia sericata (Diptera: Calliphoridae). Journal of Medical Entomology. 51(2):360-367.

Interpretive Summary: The blow fly, Lucilia sericata is an important insect species to forensic science, medicine, and agriculture. It can be both a pest species (contributing to the deterioration of lesions in free range livestock) and a beneficial insect (when used in maggot debridement technology for wound cleaning). Interest in this species has led to the development of numerous strains that require considerable manpower and rearing cost. In addition there is also the constant risk of contamination and accidental loss of strains. The study being reported in this paper details a procedure to cryopreserve and store the embryos of the blow fly in liquid nitrogen as well as a protocol to remove the embryos from storage with a high level of survival. These protocols can be used by laboratories and companies that rear this species to safeguard their valuable resources.

Technical Abstract: Embryos of Lucilia (Phaenicia) sericata (Meigen) (Diptera: Calliphoridae), the green blowfly, were successfully cryopreserved by vitrification in liquid nitrogen and stored for 8 yr. Embryos incubated at 19 deg. C for 17 h after oviposition were found to be the most appropriate stage to cryopreserve. Removal of the embryonic surface water was done using 2-propanol before the alkane treatment to permeabilize the embryo. Exposure to 2-propanol for >10 s caused necrotic tissue damage in the embryos. Among the alkanes used, hexane was found to be a superior permeabilizing solvent compared with heptane or octane, with embryo hatching rates on par with the controls. Treatment with the vitrification solution for<12 min was insufficient to vitrify the embryos. Treatment time in the solution beyond 15 min reduced embryo viability. However, the percentage of embryos vitrifying upon exposure to liquid nitrogen vapor remained constant after 12 min of treatment. Long-term storage was initiated in 2004, and the mean hatch percentage recorded then for the short-term cryopreserved embryos was 9.51%. When the long-term stored samples were retrieved in 2012, 8.47% of the embryos hatched, 66.36% larvae pupariated, and 36.96% of the pupae eclosed. Recent optimization of the technique has resulted in a hatch rate of 34.08+/- 15.5%, of which 67.5% of the larvae pupariated and 72% of the pupae eclosed to normal flies.

Last Modified: 11/22/2014
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