|CAMPBELL, JACOB - Centers For Disease Control And Prevention (CDCP) - United States|
|DOSCH, ANDREW - University Of Iowa|
|HUNT, CATHERINE - Centers For Disease Control And Prevention (CDCP) - United States|
|DOTSON, ELLEN - Centers For Disease Control And Prevention (CDCP) - United States|
|BENEDICT, MARK - Centers For Disease Control And Prevention (CDCP) - United States|
|Rinehart, Joseph - Joe|
Submitted to: Journal of Cryobiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2020
Publication Date: 4/1/2021
Citation: Campbell, J.B., Dosch, A., Hunt, C.M., Dotson, E.M., Benedict, M.Q., Rajamohan, A., Rinehart, J.P. 2021. Physiological responses to cryoprotectant treatment in an early larval stage of the malaria mosquito, Anopheles gambiae. Journal of Cryobiology. https://doi.org/10.1016/j.cryobiol.2020.12.001.
Interpretive Summary: Cryopreservation protocols have been developed for a number of insect species to protect scientifically and economically important strains against accidental loss and the harmful effects of continuous rearing. These protocols have all focused on the embryonic stage, which is widely considered the best insect stage for cryopreservation. However, some species, such as mosquitoes, have unique properties during embryonic development that make them resistant to cryopreservation. Therefore, we have investigated the feasibility of cryopreserving newly hatched larvae of the African malaria mosquito, focusing on the brief period of time before the larvae begins to feed. Initially, our studies looked at the toxicity of various cryoprotectants, which are chemicals essential to cryopreservation. We found that while several of these chemicals were reasonably non-toxic when used alone, toxicity increased when they were used as a cocktail. Next, we looked at how well these compounds entered the body of the larvae, and whether they caused water loss, both of which are also essential for successful cryopreservation. These results were more complex, suggesting that the larva’s natural ability to regulate the water content of its body also gives it the ability to regulate cryoprotectants entering and water leaving during treatment. Importantly, we also found a strong correlation between cryoprotectant toxicity and the amount of body water that was lost. While our results highlight the complexity of larval cryopreservation, we were also able to identify a specific cryoprotectant cocktail to be used in additional studies as we continue to develop a cryopreservation protocol for this critically important insect species.
Technical Abstract: The development of cryopreservation protocols for Anopheles gambiae could significantly improve research and control efforts. Cryopreservation of any A. gambiae life stage has yet to be successful. The unique properties of embryos have proven to be resistant to any practical cryoprotectant loading. Therefore, we have chosen to investigate early non-feeding first instar larvae as a potential life stage for cryopreservation. In order to determine an appropriate cryoprotective compound, larvae were treated with progressively better glass-forming cryoprotective mixtures. Toxicity evaluation in combination with calorimetry-based water content and freeze point depression assessments were used to determine the cryoprotectants that could be used for cryostorage of viable larvae. Approximately 35 – 75% of the larvae were viable after reasonably high osmotic and biochemical challenge. This study provides ample evidence for an active osmoregulatory response in the Anopheles larvae to counter the permeation of cryoprotectants from the surrounding medium. The data shows a strong correlation between the larval mortality and water content, indicating an osmoregulatory crisis in the larva due to certain cryoprotectants such as the higher concentrations of ethane diol (ED). The observations also indicate that the ability of the larvae to regulate permeation and water balance ceases at or within 20 minutes of cryoprotectant exposure, but this is strongly influenced by the treatment temperature. Among the compound cryoprotectants tested, 25% ED + 10% dimethyl sulfoxide (DMSO) and 40% ED + 0.5 M trehalose seem to present a compromise between viability, larval water content, freezing point depression, and glass forming abilities.