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United States Department of Agriculture

Agricultural Research Service


item Leopold, Roger

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 28, 2005
Publication Date: June 1, 2005
Citation: Leopold, R.A. 2005. Colony maintenance and mass-rearing: Using cold storage technology for extending the shelf-life of insects. FAO/IAEA International Conference on Area-wide Control of Insect Pests: Integrating the sterile insect and related nuclear and other techniques. May 9-13, 2005, Vienna, Austria. In: Book of Extended Synopses. pp. 100-103.

Technical Abstract: Increasing the utility and decreasing the costs of rearing insects used in research and control programs are two important concerns of scientists and managers involved in the propagation of insects. Maintaining colonies of founder and back-up strains to guard against potential losses caused by disease, work stoppage, mechanical failure, and genetic drift is a fundamental strategy used in the mass-rearing of insects but may be eliminated as a cost-cutting measure. Having the ability to stockpile beneficial insects for shipment or for release at seasonal peaks of infestation and to combat accidental pest introductions is a valuable tool not often available to project managers. Insect rearing costs can also consume large portions of research funds. Recent development of gene vectors for genetic transformation of a rapidly increasing number of species and the genomic sequencing of several highly researched species has the potential to exponentially increase the number of strains reared for research. Having the capability to place lesser-used strains or important genotypes in storage for safekeeping, would be a tremendous benefit for researchers because literally hundreds of insect strains can be maintained for a fraction of the cost as compared to continuously rearing them. For these purposes, our laboratory has been involved in the development of insect cold storage protocols for use by researchers and insectary managers. We have employed two strategies for extending insect shelf-life, cryopreservation and dormancy induction. Cryopreservation is accomplished largely through chemical/physical manipulation prior to placing the insects under liquid nitrogen storage while domancy is elicited through management of environmental factors such as photoperiod, temperature, and moisture. Successful use of either storage mechanism requires that strict attention be given to the application of these techniques at the correct age within a specific developmental stage. To date, 7 species of dipterans representing 4 families have been successfully cryopreserved as late stage embryos and, of these 7 species, protocols for Musca domestica, Cochliomyia hominivorax, Ceratitis capitata, Anastrepha suspensa, and A. ludens have been developed in our laboratory [1 - 4]. Because of inherent barriers to using conventional cryopreservation techniques that these dipteran embryos exhibit [5], development of a protocol for liquid nitrogen storage requires that each protocol be customized to accommodate the characteristics of each species. Some of the major barriers to using cryopreservation technology include: membrane impermeability, chilling sensitivity, abundance of yolk, and post storage fragility. The techniques that are used to deal with membrane impermeability involve chorion removal and membrane lipid extraction. For overcoming the chilling sensitivity of embryos equipped with abundant yolk, correct stage selection and the use of vitrification technology is required. And, to combat post storage recovery weakness, providing an adequate thawing medium and a fortified larval diet is necessary for obtaining sufficient numbers of adults to recolonize a particular species. The yield of adults after cryopreservation during their embryonic stage varies between species and also among strains within species. Recent work with 15 different screwworm strains, including wild-type, mutant, and transgenic forms, showed that hatching of cryopreserved embryos can vary between 20-78%. Evaluation of the quality of the cryopreserved insects and their progeny has been conducted under laboratory and field conditions. These tests included assessments of pupal weight, sex ratio, survival, fertility, fecundity, mating propensity and competitiveness, flight ability and endurance, and genetic diversity. Of all these observations, we found that cryopreserved screwworms had a 15% lower pupal weight which returned to the control level during the next generation. Also, there is an indication that cryopreservation of the TSL strain of the Mediterranean fruit fly may produce an altered sex ratio which probably relates to the incubation temperature of the embryos prior to treatment. With all other tests, we found no statistical differences between the cryopreserved insects or their progeny when compared to the control groups. We have also studied the induction of hibernal quiescence in parasitized and unparasitized eggs of the glassy-winged sharpshooter (GWSS), Homalodisca coagulata, as a means for stockpiling the egg parasitoids, Gonatocerus ashmeadi and G. triguttatus for subsequent release in control of this pest [6]. The rates of parasitism and emergence by G. ashmeadi decreased with the length of time that 1-day-old GWSS eggs were stored at a constant 12 deg. C and under a regime where the temperature was cycled daily between 10 and 13 deg. C . However, when unparasitized eggs were stored for 25 days, parasitism by wasps and emergence of their progeny remained statistically similar to controls only under the temperature-cycled regime. After 50 days of storage, parasitism and progeny emergence dropped 30% and 20%, respectively. Preliminary studies using moribund GWSS eggs killed by holding at 5 deg. C for 5 days indicate that parasitism and progeny emergence can be maintained near control levels after 60 days storage at 13 deg. C. After a storage period of 25 days, parasitoid emergence from the parasitized eggs stored at a constant 4.5 deg. C was significantly higher than those stored similarly at 4 deg. C. Further, the daily cycled stepwise-increasing temperature regime of 4.5, 6.0, and 7.5 deg. C changing at 8hr intervals yielded a significantly higher parasitoid emergence than a cycled regime of 4, 6, and 8 deg. C. When stored under the regime starting at 4.5 deg. C, for 10, 20 and 25 days, the emergence of wasps was 66%, 59% and 59%, respectively and was 0 after 20 days when using the regime starting at 4 deg. C.

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