Rajamohan Arun, Prasifka Jarrad R, Rinehart Joseph P
Insect Biochemistry and Genetics Research Unit, USDA-ARS-Edward T. Schafer Agricultural Research Center, 1616 Albrecht Blvd., Fargo, ND 58102, USA.
Sunflower and Plant Biology Research Unit, USDA-ARS-Edward T. Schafer Agricultural Research Center, 1616 Albrecht Blvd., Fargo, ND 58102, USA.
Insects. 2022 Oct 20;13(10):959. doi: 10.3390/insects13100959.
Embryos of the sunflower moth, (Hulst), were cryopreserved after modification to the method that was previously described for . 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° and 24 °C, and the development of the double pigment spots/eyespot and eclosion were tracked every two hours. The embryos at 24 °C showed eyespots as early as 30 h, while in the case of the embryos that were incubated at 21 °C, there was a developmental delay of approximately 20 h. The embryos at 24 °C showed peak eclosion between 55 and 70 h, and the embryos at 21 °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%.
向日葵螟（Hulst）的胚胎在对先前描述的用于[某种昆虫，此处原文未明确]的方法进行修改后进行了冷冻保存。制定该方案的工作流程包括削弱胚胎绒毛膜的方法，随后应用各种方法破坏绒毛膜下蜡层。这些步骤对于使胚胎对水和冷冻保护剂具有渗透性是必要的。最初，胚胎在21℃和24℃下孵化，每两小时跟踪双色素斑/眼点的发育和羽化情况。24℃下的胚胎最早在30小时出现眼点，而在21℃下孵化的胚胎则有大约20小时的发育延迟。24℃下的胚胎在55至70小时之间出现羽化高峰，21℃下的胚胎在发育80至100小时之间羽化。估计这个范围对于胚胎冷冻保存方案开发的阶段选择和处理开始至关重要。在任一发育温度下，对照孵化率均>90%，而次氯酸钠、2-丙醇和基于烷烃的处理将胚胎孵化率降低至<10%。因此，使用改良的表面活性剂-次氯酸盐混合物来破坏绒毛膜并溶解疏水脂质层。使用染料摄取法进行的水渗透性评估表明，聚山梨醇酯80与次氯酸钠单独组合能够像次氯酸盐-烷烃顺序处理一样有效地使胚胎通透，但孵化率明显更高。由乙二醇和海藻糖组成的玻璃化介质用于使胚胎脱水并加载冷冻保护剂。玻璃化后的中位孵化率为10%，最高为23%。
