High-Humidity Incubation Improves Post-Microinjection Viability and Larval Performance of CRISPR/Cas9-Edited...

Gene editing technologies are playing an increasingly important role in the study of insect gene functions. Appropriate incubation humidity helps improve the hatching rate of insect eggs. However, to date, no study has evaluated the impact of humidity on the hatching rate of gene-edited eggs or the subsequent development of the larvae. Using the cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) as a model, we investigated the weight and hatching rate of microinjected eggs under different humidity conditions. We determined the larval development time and pupal weight and calculated the larval gene editing efficiency. The results showed that post-microinjection incubation under high humidity (RH = 95%) had a significant influence on CRISPR/Cas9 gene-edited eggs. The key research results indicate that, compared with the incubation environment with RH ≤ 80%, after 48 h of incubation under 95% RH, the hatching rate of H. armigera eggs increased by more than 27.5%, with higher egg saturation and greater egg weight; meanwhile, the larval developmental duration was shorter and the pupal weight was also greater. These results establish a humidity-regulated developmental recovery protocol for gene-edited insects, which provides theoretical and practical support for optimizing post-microinjection recovery processes in gene-edited insects.