Submitted to: Cryobiology
Publication Type: Abstract Only
Publication Acceptance Date: 7/15/2003
Publication Date: 12/1/2003
Citation: Leopold, R.A., Rajamohan, A., Shelly, T.E. 2003. Development and results of quality assurance testing for mass-reared and laboratory-colonized insects after embryo cryopreservation. Abstracts/Cryobiology. 47(3):270.
Technical Abstract: Developing cold storage protocols to supplement rearing regimes for factory- or laboratory-propagated insects can be useful or even a required strategy to ensure that production is economical, reliable, and results in quality insects. However, there is widespread concern by first time users of insects having experienced a 'cold storage bottleneck' that irreparable damage or change may have occurred. Resistance by insect rearing personnel to incorporation of a new technology such as insect embryo cryopreservation into an established rearing regime is especially common. To allay these concerns, we have devised and conducted a number of quality assurance tests on 4 species of insects that are extensively propagated for release in area-wide autocidal control programs. The insects tested for fitness after cryopreservation via vitrification were: the Caribbean (CBFF), Mexican (MXFF), and Mediterranean (MDFF) fruit flies and the New World screwworm. For the CBFF, lab determinations were made on fertility and fecundity. Adult progeny from parents cryopreserved as embryos were paired with untreated flies. The number of eggs collected from the mating pairs and the larval hatching was not significantly different from the control levels (P > 0.01; n = 10/sample). Male mating propensity and flight mill endurance of male and female MXFF cryopreserved as embryos was evaluated under laboratory conditions and found to be the same as that of the controls (P > 0.0001, n = 30 & P > 0.5; n = 37). Preliminary studies using RAPD analysis for measurement of a possible change in genetic diversity of MXFF recolonized after cryopreservation, suggest no overall bias in RAPD marker frequencies when compared to untreated parent colony flies. Progeny (G3) from cryopreserved factory-reared MDFF were field-tested for survival, mating competitiveness, and fecundity. We found no significant differences in these fitness parameters when compared to those of a wild-type control strain (P > 0.05; n = 100/sample). Further, lab comparisons on flight ability of newly emerged MDFF adults showed that the cryopreserved line exceeded that of the wild-type (P < 0.01; n = 100). Screwworms recovered from liquid nitrogen storage had lower pupal weights and pupation and adult emergence rates than the untreated control flies (P < 0.05; n = 200-600/sample) but the sex ratios were similar. However, by the next generation these differences were no longer present. In summary, we have tested 4 species of flies cryopreserved as embryos or as progeny of cryopreserved parents for a number of indicators of fitness or quality. Based on the results, there is little to suggest that permanent damage or change occurs when using an insect cryopreservation protocol such as been developed in our laboratory (Leopold et al. Ann. Entomol. Soc. Amer. 94, 695-701, 2001; Rajamohan et al. Cryo-Lett. 24, 125-132, 2003).