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Effects of Embryonic Development Stage And Cryoprotective Agents on Galleria Mellonella Embryo Cryopreservation

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BACKGROUND: The increased research use of the honeycomb pest Galleria mellonella has created the need for cryopreservation. The diverse characteristics of G. mellonella embryos at different stages may affect embryo survival upon cryopreservation due to differential dehydration, cold resistance, cryoprotectant toxicity and permeability. OBJECTIVE: The study aimed to determine the ability of G. mellonella to survive cryogenic condition in the early and late embryonic developmental stages (24h and 75h post–oviposition, PO). MATERIALS AND METHODS: A modified protocol was used to select the proper embryonic stage by comparing the early and late embryonic stages under two procedures of dechorionation-permeabilization (D/P) dilutions. The embryos at the early stage were used for testing the toxicity and viscosity effects of various cryoprotective agents. Various cryoprotectant treatments for improving the hatch rate were studied. RESULTS: The survival rates of embryos at the early stages (24h PO) were 51.5% and 69.5% respectively after cryoprotectant loading in 12% ethylene glycol (EG) for 30 min and dehydration in vitrification solution for for 10min. These survival rates of embryos at the late stages (75h PO), however, decreased to 22.9% and 34.0%, respectively. D/P treatment with Tween 20 and Tween 80 (2:6) slightly improved the survival of embryos at the 24h PO stage after immersion in liquid nitrogen (LN) from 0.1% to 0.6%. The increased exposure time in dehydration treatment from 14 to 30min in two vitrification solutions, EG and methanol (MeOH), slightly increased the survival rate of cryopreserved embryos from 0% and 0.3% to 1.1% and 1.4%, respectively, while the vitrification solution containing dimethyl sulfoxide (Me2SO or DMSO) slightly decreased the hatch rate from 1% to 0.6%. CONCLUSION: The survival rate of G. mellonella embryos after cryopreservation was affected by the oviposition period, eggshell permeability to cryoprotectant, cryoprotectant type, as well as their concentration and exposure times.
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Keywords: CRYOPRESERVATION; CRYOPROTECTANT; EMBRYOGENESIS; GALLERIA MELLONELLA; LEPIDOPTERA

Document Type: Research Article

Publication date: July 1, 2018

More about this publication?
  • CryoLetters is a bimonthly international journal for low temperature sciences, including cryobiology, cryopreservation or vitrification of cells and tissues, chemical and physical aspects of freezing and drying, and studies involving ecology of cold environments, and cold adaptation

    The journal publishes original research reports, authoritative reviews, technical developments and commissioned book reviews of studies of the effects produced by low temperatures on a wide variety of scientific and technical processes, or those involving low temperature techniques in the investigation of physical, chemical, biological and ecological problems.

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