Location: Tropical Pest Genetics and Molecular Biology Research Unit
Project Number: 2040-30400-003-012-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jun 1, 2026
End Date: Dec 31, 2027
Objective:
We will adopt the published cryopreservation protocols for our in-house Mexfly and Oriental fruit fly in a short-term (less than 6 months) storage. Previous cryopreservation protocol has been established for Medflies and Mexfly, however, the strain, genetic background, and/or the environment can affect cryopreservation efficiency. We will use the published protocols to guide us in developing a standard operating procedure for our in-house fruit flies currently used or will be used in SIT programs for population control. Short-term cryopreservation will be assessed to optimize the protocol by assessing the hatch percentage of recovered embryos.
Approach:
Using the published protocols as a guide (Augustinos et al. 2016, Rajamohan and Leopold 2007), embryos will be collected from Oriental fruit fly and Mexfly available in-house and reared at a range of 18-25 C to slow development and facilitate selection of optimal developmental stage for vitrification. Embryo development phenotype determined for optimal vitrification in other strains of fruit flies was when embryos showed a helical shaped gut (Augustinos et al. 2016, Rajamohan and Leopold 2007). However, we will assess this and other developmental embryonic stages for the optimal stages of our in-house fruit flies and determine the time and temperature that will be most efficient in achieving the optimal stage. Subsequently, embryos will be dechorionated using sodium hypochlorite, permeabilized using hexane, and loaded with cryoprotectant using different cocktail mixes of ethylene glycol, polyethylene glycol-8000, and trehalose. Concentrations of sodium hypochlorite, hexane, and cryoprotectant mixes will be optimized for our in-house strains. Embryos will then be vitrified on a polycarbonate membrane or other tested vehicle (e.g., cryovial) and stored in a liquid nitrogen dewar for a short-term (less than 6 months). Embryos will be recovered from cryopreservation, and the quality control measures will be assessed. The initial quality control measure will focus on hatch percentages to screen the cryopreservation methodologies.