Project Number: 3060-21220-032-011-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Jul 1, 2021
End Date: Jun 30, 2023
This project has two main components. The first is the cryopreservation of non-feeding 1st instar mosquito larvae. Although the difficulties associated with the cryopreservation of mosquito eggs is well documented, the cryopreservation of 1st instar larvae is no less daunting. Our team has focused on the non-feeding stage (up to 90 minutes after hatching) to reduce the likelihood of inoculative freezing initiated by gut contents that could lead to a failure to vitrify. Our cryoprotectant toxicity showed promise, with permeating cryoprotectants proving to be mostly non-toxic, and both permeating-permeating and permeating-nonpermeating cryoprotectant cocktails showing increased toxicity. Reducing the temperature during cryoprotectant loading resulted in a significant improvement in survival, which is consistent with our insect embryonic cryopreservation studies. However, whole body differential scanning calorimetry (DSC) indicated that cryoprotectant levels inside the larvae was either not sufficiently high or differentially compartmentalized within the larval structures, and that the larval water content was surprisingly dynamic, suggesting that an active osmoregulatory response was involved, leading to a reduction in the permeation of cryoprotectants from the surrounding medium. Hence, further studies will focus on determining the magnitude to which compartmentalization is an issue and using different techniques to increase cryoprotectant levels within the larvae. Our second component in the cryopreservation of mosquito spermatozoa. Although an optimal germplasm cryopreservation program includes both oocyte and spermatozoa cryopreservation, programs only employing spermatozoa cryopreservation can also be successful. For instance, the sole cryopreservation technique for the honey bee, and the only available cryopreservation technique for many avian species is spermatozoa cryopreservation. Additionally, the spermatozoa of most species are highly amenable to time-tested conventional slow freezing cryopreservation techniques, and pure lineages can be re-established from cryopreserved spermatozoa in as little as six generations. Using 10% DMSO as a cryoprotectant, and with a freezing rate of 0.25°C/min, we were able to develop a slow freezing protocol for Anopheles gambiae whole testis that resulted in sperm viability after recovery from liquid nitrogen that ranged from 70% to nearly 95%. Although promising, much work remains to be done to develop this technique into a reliable, transferrable technology.
We have two specific goals to improve the likelihood of successful larval cryopreservation. Compartmentalization analysis. The mosquito larva is a uniquely complex structure for cryopreservation. Although cryoprotectants may enter the body, non-uniform inter compartmental distribution could lead to problems with vitrification. To assess the likelihood of this issue, we will conduct cryoprotectant loading as reported before, and then conduct cryo-microscopy assisted differential scanning calorimetry using newly acquired equipment. This will allow us to determine the degree to which compartmentalization is occurring, the concentration of cryoprotectants in different areas of the body, and whether ice nucleation events are a complicating issue. Increasing permeating cryoprotectant concentrations by desiccation. Another potential strategy to increase internal cryoprotectant concentrations is by using the fact that the mosquito larva is uniquely susceptible to desiccation. While preliminary studies suggest that simple air drying is problematic, we will attempt to standardize the process. Initial studies will involve streams of air with varying flow rates and humidity to identify a reliable combination that increases cryoprotectant concentrations enough to support vitrification without desiccating so much that there is an increase in mortality. Subsequent studies will involve floatation-dehydration, which has been used successfully on other dipteran species. We have three specific goals for improving Anopheles spermatozoa cryopreservation, as follows. Optimization of cryoprotectant and extender mediums. While we have demonstrated that 10% DMSO can be used to cryopreserve mosquito spermatozoa, whether this is optimal for reproducible survival and optimal sperm quality remains to be elucidated. Hence, we will be testing the efficacy of a range of cryoprotectants in a range of concentrations to improve overall cryopreservation success. Additionally, our group has demonstrated that the ionic composition of the extender medium used prior to and after cryopreservation can have a marked effect on success in other species, and we expect similar results for mosquitos as well. Hence, extender medium with a range of concentrations for specific ions will also be investigated. Slow freezing vs vitrification. Although spermatozoa are uniquely amenable to slow freezing techniques, downstream negative consequences, such as residual cryoprotectants in semen leading to diminished maternal fitness after artificial insemination are of concern. Hence, we will also pursue vitrification techniques as an alternative to slow freezing. This has shown promise for other insect species in our laboratory. Comparison of different strains and species. Although differences amongst An. gambiae strains and between different species of Anopheline mosquitoes is less of a concern with spermatozoa cryopreservation, a comparative study should still be conducted prior to widespread use.