Vitrification of Lepidopteran Embryos-A Simple Protocol to Cryopreserve the Embryos of the Sunflower Moth, Homoeosoma electellum

Simple Summary Since originally being described for fruit flies in 1990, insect embryo cryopreservation protocols have been developed for several fly species and have been used to preserve several important fly species and strains. However, the development of similar protocols for other types of insects has not progressed as rapidly. For instance, only three species of moths have been successfully cryopreserved, and the technique has not been widely adopted for any of these species. This is mainly due to complications associated with the outer egg coatings surrounding the developing moth embryo, which are substantially different from what surrounds a fly embryo. To expand the usefulness of this technique for moths, we have developed a cryopreservation protocol for the sunflower moth, a key pest of cultivated sunflower in the southern Great Plains of the United States. Our final protocol resulted in 23% of embryos hatching after cryopreservation, with 60% surviving to the adult stage. While designing this protocol, we also developed a novel method to quantitatively assess the ability of water to leave the egg after treatments, which is critically important to successful cryopreservation. Embryos of the sunflower moth, Homoeosoma electellum (Hulst), were cryopreserved after modification to the method that was previously described for Pectinophora gossipiella. The workflow to develop the protocol consisted of methods to weaken the embryonic chorion followed by the application of various methods to disrupt the sub-chorionic wax layer. These steps were necessary to render the embryos permeable to water and cryoprotectants. Initially, the embryos were incubated at 21 degrees and 24 degrees C, and the development of the double pigment spots/eyespot and eclosion were tracked every two hours. The embryos at 24 degrees C showed eyespots as early as 30 h, while in the case of the embryos that were incubated at 21 degrees C, there was a developmental delay of approximately 20 h. The embryos at 24 degrees C showed peak eclosion between 55 and 70 h, and the embryos at 21 degrees C eclosed between 80 and 100 h of development. Estimating this range is crucial for the purposes of stage selection and treatment initiation for cryopreservation protocol development for the embryos. The control hatch percentage at either developmental temperature was >90%, and the sodium hypochloride, 2-propanol and alkane-based treatments reduced the embryo hatchability to <10%. Hence, a modified surfactant-hypochlorite mixture-was used to destabilize the chorion and solubilize the hydrophobic lipid layers. Water permeability assessments using the dye-uptake method show that polysorbate 80 in combination with sodium hypochlorite alone is capable of permeabilizing the embryo as efficiently as sequential hypochlorite-alkane treatments, but with significantly higher hatch rates. A vitrification medium consisting of ethane diol and trehalose was used to dehydrate and load the embryos with the cryoprotective agent. The median hatch rates after vitrification were 10%, and maximum was 23%.