DEVELOPMENT OF COLD STORAGE TECHNOLOGY FOR MASS-REARED AND LABORATORY-COLONIZED INSECTS
Location: Insect Genetics and Biochemistry Research
Title: Quality Testing of Three Species of Tephritid Fruit Flies After Embryo Cryopreservation
Submitted to: Annals of the Entomological Society of America
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 13, 2009
Publication Date: March 1, 2010
Citation: Leopold, R.A., Rajamohan, A., Shelly, T.E., Handler, A.M. 2010. Quality Testing of Three Species of Tephritid Fruit Flies After Embryo Cryopreservation. Annals of the Entomological Society of America. 103(2):264-272.
Interpretive Summary: This study was conducted to determine whether the process of cryopreservation had detrimental effects on the physiological, behavioral or genetic aspects that have been deemed important to insects that are used for research or control purposes. Cryopreservation is a process whereby cells and organisms are treated in a manner that allows them to be stored in the cold using liquid nitrogen for long periods of time. Cryopreservation of insects has been promoted as a possible solution for the often experienced loss of quality factors in insects that are maintained over repeated generations under laboratory or factory-like conditions. These quality factors include adult emergence, longevity, mating ability, fecundity, fertility, flight ability, and genetic diversity. Three species of insects, the Mexican, Mediterranean, and Caribbean fruit flies, were used to test the various quality factors under laboratory and field conditions after they had been cryopreserved as embryos. For almost all of the tests, we found no adverse effects had been conveyed to flies that had been cryopreserved or to projeny of flies that had been cryopreserved. Overall, the difference gained from the unfed control level longevity test using Mexican fruit flies was not of major import when weighed against all the other positive results gained from this study. It was clearly evident that embryo cryopreservation had little effect on the quality factors tested and that it should be strongly considered for long term programs rearing insects in the laboratory and in mass-production facilities.
This study evaluates characteristics commonly used to define insect quality or fitness by using a complement of three species of tephritid fruit flies obtained from cryopreserved embryos. The Mexican, Anastrepah ludens, Caribbean, A. suspense, and Mediterranean, Certatitis capitata, fruit flies were used to assess embryo to adult emergence and adult longevity, flight ability, mating ability, fecundity, and genetic variability following cryopreservation. With the 3 species tested, embryo survival was reduced by 50-70% while adult eclosion appeared unaffected by cryogenic treatment. Laboratory cage survival of cryopreserved A. ludens paralleled that of controls when tested with or without food and water post treatment. With C. capitata, field cage survival was also similar when the adult progeny of cryopreserved parents was compared to that of untreated flies of the same age. Assessment of flight ability of cryopreserved A. ludens over a 19 d period using a flight mill showed no statistical difference when compared to the untreated groups over the same time perios. Flight ability within field cages for newly emerged progeny of cryopreserved C. capitata also mirrored that of the controls. Observed matings occurring with laboratory cages containing equal numbers of A. ludens males and females did not differ from comparable control groups. Further, male progeny obtained from cryopreserved C. capitata parents competed equally with untreated males for mates while housed in field cages. A laboratory analysis of fertility and fecundity of A. suspense revealed that males mated with control females were unaffected by cryopreservation as embryos while cryopreserved females exhibited a significantly reduced fecundity when mated with control males. The fecundity of C. capitata progeny of cryopreserved parents also did not differ from control levels while caged under laboratory conditions. A RAPD assay of the gentic diversity of A. ludens comparing cryopreserved males with control males showed that the coefficient of similarity was ~85%. This study indicates that embryo cryopreservation had little or no affect on the reproduction, longevity and flight of the species tested and an be used to support maintenance of insect stocks and control programs supported by the mass-rearing process.