Submitted to: CryoLetters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/2/2003
Publication Date: 5/20/2003
Citation: RAJAMOHAN,A., LEOPOLD,R.A., WANG,W.B., HARRIS,M., MCCOMBS,S.D., PEABODY,N.C., FISHER,K., CRYOPRESERVATION OF MEDITERRANEAN FRUIT FLY EMBRYOS, CRYOLETTERS 24:125-132, 2003.
Interpretive Summary: The Mediterranean fruit fly or medfly, Ceratitis capitata, is one of the most serious pests of fruit crops world-wide. Control programs involve the use of pesticide sprays, poisoned baits and area-wide sterile-male releases. The sterile insect technique (SIT) involves mass-rearing large numbers of flies, which are sexually sterilized and released to reproductively compete with the wild population. Even though the technique has been very successful, the rearing facilities may experience reduced production, which can be caused by various factors such as diseases, mechanical failures and inbreeding. An approach to alleviate potential production problems is to maintain a cryopreserved back-up store of insects, which could be revived to renew or revitalize the current production strain that is under duress. Another potential use of insect cryopreservation is in research programs where the economics of maintaining large numbers of genetic strains becomes burdensome. To this end, we have developed an embryo cryopreservation procedure for the bisexual strain of medfly that yields about 34% normal adults after storage in liquid nitrogen and a larval development in a diet having an agar base. Post cryopreservation recovery using diets having either a corncob grit or wheat bran base were not successful. However, these latter diets can be used for subsequent generations after the initial post cryopreservation recovery.
Technical Abstract: In this paper we present a procedure to cryopreserve the embryos of the tephritid, Mediterranean fruit fly (Ceratitis capitata) by vitrification. Developmental stages between 24 and 32 hours after oviposition were examined for tolerance to cryopreservation. Embryos, 27-hr-old and incubated at 29 deg. C, were found to be at the most suitable stage for treatment. Effects of the previtrification steps of our protocol, dechorionation, permeabilization, cryoprotectant loading, and dehydration, on survival to hatching were also assessed. Dechorionation did not affect viability, while isopropanol and a hexane treatment used in the permeabilization step of the protocol reduced hatching by about 15%. This reduction was dependant on the amount of isopropyl alcohol carried over into the hexane rinse. The remaining previtrification steps reduced hatching by an additional 10%. After optimization of the procedure, normalized hatching was 44% after vitrification in liquid nitrogen vapor followed by storage under liquid nitrogen for a test period of 7 days. Post cryopreservation larval diets containing wheat bran, corn cob grits, or agar as the base were examined for survival to pupation and emergence. A yield of 34% egg to adult emergence was obtained when the agar-based diet was used for rearing larvae that had experienced cryopreservation during the embryonic stage.