A method for cryopreservation of ovaries of the ladybird beetle, Harmonia axyridis

Abstract

The development of genome editing techniques has allowed the generation of tremendous genetic diversity, even in non-model insects. The major obstacle for maintenance of genetically diverse stocks in non-model insects is the need for constant rearing in the laboratory, which is labor-intensive and time-consuming. The maintenance of insect colonies in the laboratory is further complicated by risk factors such as disease contamination, human error, and genetic changes by natural mutations that can lead to the loss of desirable genotypes. To avoid these risk factors, cryopreservation is the most desirable option. Here, we present a method we developed for cryopreservation of ovary from the multicolored Asian ladybird beetle, Harmonia axyridis, adapted from techniques for ovary cyropresevation in the silkworm, Bombyx mori. We used last (4th) instar beetle larvae as both donors and recipients for the cryopreserved ovaries. The best and average success rates of cryopreservation were 40% and 26%, respectively, based on the percentage of beetles receiving cryopreserved ovary transplants that subsequently laid viable eggs. This success rate is much higher than that reported for B. mori using last instar larvae. The 26% average success rate is sufficient for the technique to be of practical use for maintaining genetically diverse lines of H. axyridis, and reduces the laborious tasks required for constant maintenance of numerous colonies. This is the first reported success in ovary cryopreservation for a small non-model insect. The techniques that we developed should also be useful for ovary cryopreservation in other small non-model insects.