Whole testicular organ and seminal cryopreservation in the malarial mosquito Anopheles gambiae (Diptera: Culicidae)

Abstract

Anopheles gambiae (Giles) is a medically important mosquito species and a vector of the malaria Plasmodium parasite. There have been many advancements in vector control research that involves genetic manipulation of the mosquito. Malaria vector research requires rearing continuous mosquito colonies that are costly and carry risks to maintain. Therefore, we studied the feasibility of germplasm cryopreservation as a way to maintain genetic background as well as restore mosquito colonies. One part of germplasm cryopreservation is the cryopreservation of the whole germinal organ such as the testis as this would preserve half the genome. The aim of this study was to develop a simple method to cryopreserve whole testicular-seminal vesicle complex from which one could derive both the viable spermatozoa as well as primordial and stem cells from male mosquitos. This is the first study which demonstrates the successful cryopreservation of a whole organ in an insect system. In this study, various cryopreservation protocols were tested to determine which resulted in the highest testicular cell survival. The methods tested included different freezing rates, different incubation protocols, and use of different non-permeating cryoprotectants. The cooling rate that was found to have the highest survival was cooling to −40 °C at a rate of 1 °C min−1 followed by a plunge into liquid nitrogen, although there was little difference in survival based on cooling rate.