Mythimna separata (Walker) (Lepidoptera: Noctuidae) is one of the most destructive phytophagous pests. Here we present detailed instructions to achieve successful application of CRISPR/Cas9‐mediated genome editing on M. separata. We optimized procedures, including egg collection and incubation. We further successfully conducted microinjection on the washed eggs by applying water on the egg surface. We then evaluated the effects of injection timing on both egg viability and genome editing efficiency using NPC1b as the target gene. We observed that Cas9 protein injection had significantly less effect on the viability of the eggs at the earlier stage (<2 h old) than at later stages, although no significant effect was observed on the postembryonic development between different egg ages. All larvae were edited when the eggs were <6 h old, and the editing efficiency decreased when older eggs were used. Moreover, mutants were detected among G1 progenies, which demonstrated that the mutation generated by the Cas9/sgRNAs system on M. separata was heritable. As far as we know, this is the first report of CRISPR/Cas9 for this important pest. The successful application of genome editing on M. separata is essential to understanding its biology and developing novel control methods.

Plant triterpenoids play a critical role in plant resistance against Phytophthora infestans de Bary, the causal pathogen of potato and tomato late blight. However, different triterpenoids could have contrasting functions on plant resistance against P. infestans. In this study, we targeted the key biosynthetic gene of all plant triterpenoids, SQUALENE SYNTHASE (SQS), to examine the function of this gene in plant–P. infestans interactions. A post-inoculation, time-course gene expression analysis revealed that SQS expression was induced in Nicotiana benthamiana but was transiently suppressed in Solanum lycopersicum. Consistent with the host-specific changes in SQS expression, concentrations of major triterpenoid compounds were only induced in S. lycopersicum. A stable overexpression of SQS in N. benthamiana reduced plant resistance against P. infestans and induced the hyperaccumulation of stigmasterol. A comparative transcriptomics analysis of the transgenic lines showed that diverse plant physiological processes were influenced by SQS overexpression, suggesting that phytosterol content regulation may not be the sole mechanism through which SQS promotes plant susceptibility towards P. infestans. This study provides experimental evidence for the host-specific transcriptional regulation and function of SQS in plant interactions with P. infestans, offering a novel perspective in examining the quantitative disease resistance against late blight.

Insects are sterol auxotrophs and typically obtain sterols from food. However, the sterol demand and metabolic capacity vary greatly among species, even for closely related species. The low survival of many insects on atypical sterols, such as cholestanol and cholestanone, raises the possibility of using sterol-modified plants to control insect herbivore pests. In this study, we evaluated two devastating migratory crop pests, Mythimna separata and Spodoptera frugiperda, in response to atypical sterols and explored the reasons that caused the divergences in sterol nutritional biology between them. Contrary to M. separata, S. frugiperda had unexpectedly high survival on cholestanone, and nearly 80% of the individuals pupated. Comparative studies, including insect response to multiple diets and larval body sterol/steroids analysis, were performed to explain their differences in cholestanone usage. Our results showed that, in comparison to M. separata, the superiority of S. frugiperda on cholestanone can be attributed to its higher efficiency of converting ketone into available stanol and its lower demand for sterols, which resulted in a better survival when cholesterol was unavailable. This research will help us to better understand insect sterol nutritional biology and the potential of using atypical sterols to control herbivorous insect pests.