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Published on: February 2, 2021
Cryoprotectant toxicity and hypothermic sensitivity among Anopheles larvae
Jenny E Nesbitt1, Anisa Swei1, Catherine Hunt2
1BioMEMS Resource Center, Center for Engineering in Medicine, & Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
Abstract:
Laboratory rearing of mosquitoes is commonly practiced by researchers studying mosquito-borne infectious diseases and vector control methods. In the absence of cryopreservation methods to stabilize unique or genetically modified strains, mosquito lines must be continuously maintained, a laborious process that risks selection effects, contamination, and genetic drift. Towards the development of a cryopreservation protocol, several commonly used cryoprotectants were systematically characterized here both individually and as cocktails. Among first instar, feeding-stage An. gambiae and An. stephensi larvae, cryoprotectant toxicity followed the order of dimethyl sulfoxide > ethylene glycol > methanol. The resulting LD50 values were used as the basis for the development of cryoprotectant cocktail solutions, where formulation optimization was streamlined using Taguchi methods of experimental design. Sensitivity to hypothermia was further evaluated to determine the feasibility of cryoprotectant loading at reduced temperatures and slow cooling approaches to cryopreservation. The information described here contributes to the knowledge base necessary to inform the development of a cryopreservation protocol for Anopheles larvae.
Insights
Developing a cryopreservation protocol for mosquito larvae is crucial for disease research. This study characterized cryoprotectant toxicity and optimized solutions for Anopheles gambiae and Anopheles stephensi, paving the way for stable mosquito lines.
Area of Science:
- Vector Biology
- Entomology
- Cryobiology
Background:
- Laboratory mosquito colonies are essential for studying mosquito-borne diseases and developing control strategies.
- Maintaining live mosquito colonies is labor-intensive and susceptible to genetic drift and contamination.
- Current lack of cryopreservation methods necessitates continuous colony maintenance.
Purpose of the Study:
- To systematically characterize commonly used cryoprotectants for Anopheles larvae.
- To develop and optimize cryoprotectant cocktails for mosquito larval cryopreservation.
- To assess hypothermia sensitivity for cryopreservation feasibility.
Main Methods:
- Systematic characterization of individual cryoprotectants (dimethyl sulfoxide, ethylene glycol, methanol) and their toxicity.
- Determination of lethal dose 50 (LD50) values for Anopheles gambiae and Anopheles stephensi larvae.
- Optimization of cryoprotectant cocktail formulations using Taguchi methods and evaluation of hypothermia sensitivity.
Main Results:
- Cryoprotectant toxicity in first instar larvae followed the order: dimethyl sulfoxide > ethylene glycol > methanol.
- LD50 values informed the development of cryoprotectant cocktail solutions.
- Sensitivity to hypothermia was assessed to guide cooling protocols.
Conclusions:
- This research provides foundational data for developing a cryopreservation protocol for Anopheles larvae.
- Optimized cryoprotectant formulations and understanding of hypothermia sensitivity are key steps.
- Successful cryopreservation would significantly benefit mosquito-borne disease research and vector control efforts.
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