Cryopreservation of Anopheles stephensi embryos

Abstract

The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human infectious diseases by conserving stocks, new isolates, lab-bred strains, and transgenic lines that currently require continuous life cycle maintenance. Efforts over several decades to develop a method for cryopreservation have, until now, been fruitless: we describe here a method for the cryopreservation of Anopheles stephensi embryos yielding hatch rates of ~ 25%, stable for > 5 years. Hatched larvae developed into fertile, fecund adults and blood-fed females, produced fully viable second generation eggs, that could be infected with Plasmodium falciparum at high intensities. The key components of the cryopreservation method are: embryos at 15-30 min post oviposition, two incubation steps in 100% deuterated methanol at - 7 °C and - 14.5 °C, and rapid cooling. Eggs are recovered by rapid warming with concomitant dilution of cryoprotectant. Eggs of genetically modified A. stephensi and of A. gambiae were also successfully cryopreserved. This enabling methodology will allow long-term conservation of mosquitoes as well as acceleration of genetic studies and facilitation of mass storage of anopheline mosquitoes for release programs.

Figure 1

Parameters for oviposition and incubation in cryoprotectant additive (CPA: methanol) for A. stephensi egg cryopreservation. (a) Survival (percent hatch) of eggs following incubation in 100% methanol for 15 min at temperatures between 0 and − 13 °C without subsequent cooling and with recovery into water at 23 °C. (b) Survival (percent hatch) of eggs following incubation in 100% methanol for periods of 7–45 min at − 11 °C and subsequent dilution into water at 23 °C. Eggs directly warmed/diluted from − 11 °C without being rapidly cooled by plunging into LN₂ (solid line); eggs incubated at − 11 °C and subsequently plunged into LN₂ before warming/dilution (dashed line). (c) Optimization of the incubation temperature for the first CPA incubation step with the second incubation step held constant at − 14.5 °C for 15 min. (d) Optimization of the incubation temperature for the second CPA incubation step with the first incubation step held constant at − 7 °C for 6.5 min. (e) Effect of oviposition time on survival: eggs were oviposited onto water in Petri dishes for 15 min, harvested and then held an additional 5–75 min before adding to CPA (single incubation at − 11 °C for 15 min). The data points represent the age of the oldest eggs in each sample (20–90 min old).