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Cryopreservation of Nasonia vitripennis Embryos

机译：低温保藏Nasonia vitripennis胚胎

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Nasonia vitripennis is a small parasitoid wasp that lays eggs in the pupa of pest flies, and has been used as a model insect for genetics. Since Nasonia embryos have not been cryopreserved successfully, we tried to cryopreserve the embryos. Female wasps were allowed to lay eggs in fly pupae for 3 h, and the oocytes were cultured for 20 h at 24℃ to obtain parthenogenetically developed embryos. To assess the survival of embryos after various experimental treatments, they were cultured for 20 h at 24℃, and those developed to larvae were considered survived. We examined the suitable condition for removing wax layers on the embryo, the permeability of embryos just after being freed from wax layers (wax-free embryos) to water and cryoprotectants, the toxicity of cryoprotectants and of an ethylene glycol (EG) -based vitrification solution (EFS40) to wax-free embryos, and finally whether wax-free embryos can survive after cryopreservation by vitrification using 10% EG solution and EFS40. Wax layers of the embryo could be removed most efficiently when they were treated with iso-propanol for 60 sec and then with hexane for 150 sec. From the volume change of wax-free embryos, it was shown that water, DMSO, and EG were able to move across the plasma membrane of the embryos. It was supposed that water diffused out rapidly, and EG permeated the embryos faster than DMSO. Wax-free embryos were highly resistant to the toxicity to 10% EG solution but were able to survive in EFS40 only a few minutes at 25℃. After vitrification with EFS40, about 50% of embryos survived but none of them hatched. The cryopreservation of Nasonia embryos would be realized if the chorion can be removed from the embryos safely.
机译：Nasonia vitripennis是一种小的寄生蜂，在害虫蝇pest中产卵，已被用作遗传学的典范昆虫。由于Nasonia胚胎尚未成功冷冻保存，因此我们尝试冷冻保存胚胎。让雌性黄蜂在蝇p中产卵3 h，并将卵母细胞在24℃下培养20 h，以获得单性生殖发育的胚胎。为了评估各种实验处理后的胚胎存活，将它们在24℃下培养20 h，并将发育为幼虫的胚视为存活。我们研究了去除胚胎上蜡层的合适条件，从蜡层释放出来的胚胎（无蜡的胚胎）对水和冷冻保护剂的渗透性，冷冻保护剂和基于乙二醇（EG）的玻璃化的毒性溶液（EFS40）处理不含蜡的胚胎，最后使用10％EG溶液和EFS40将玻璃冷冻保存后，不含蜡的胚胎能否存活。当用异丙醇处理60秒，然后用己烷处理150秒时，可以最有效地去除胚胎的蜡层。从不含蜡的胚胎的体积变化可以看出，水，DMSO和EG能够穿过胚胎的质膜。可以认为，水迅速扩散，而EG比DMSO更快地渗透到胚胎中。无蜡的胚胎对10％EG溶液的毒性具有很高的抵抗力，但在25℃的EFS40中仅能存活几分钟。用EFS40玻璃化后，大约50％的胚胎存活了下来，但没有一个孵化。如果绒毛膜可以安全地从胚胎中取出，则可以实现对Nasonia胚胎的冷冻保存。

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