RNAi and CRISPR/Cas9 as functional genomics tools in the neotropical stink bug, Euschistus heros

Understanding the biology of insect pests is an important step towards developing appropriate control strategies. In this study, a CRISPR/Cas9 gene knockout work flow was established for the first time and was together with RNAi used as tools to study gene functions in the Neotropical stink bug, Euschistus heros. RNAi was first employed to study the function of three genes, abnormal wing disc (awd), tyrosine hydroxylase (th) and yellow (yel). Targeting awd and th resulted in distinct malformed phenotypes such as a deformed wing or a lighter cuticle pigmentation/defects in cuticle sclerotization, respectively. However, no distinct phenotype was observed for yel. To further investigate the function of yel, a CRISPR/Cas9 gene editing protocol was developed for E. heros. A total of 719 eggs were microinjected with single-guide (sgRNA) and Cas9 and total of six insects hatched. Out of these six nymphs, one insect showed mutation in yel but no clear phenotype was visible. Although, we were unable to generate insects with a distinct phenotype for yel, a successful gene editing workflow was established to complement RNAi for future functional gene studies in E. heros. Additionally, we provided recommendations to improve the established gene editing workflow. The Neotropical brown stink bug, Euschistus heros, is one of the most important stink bug pests in leguminous plants in South America. RNAi and CRISPR/Cas9 are important and useful tools in functional genomics, as well as in the future development of new integrated pest management strategies. Here, we explore the use of these technologies as complementing functional genomic tools in E. heros. Three genes, abnormal wing disc (awd), tyrosine hydroxylase (th) and yellow (yel), known to be involved in wing development (awd) and the melanin pathway (th and yel) in other insects, were chosen to be evaluated using RNAi and CRISPR/Cas9 as tools. First, the genes were functionally characterized using RNAi knockdown technology. The expected phenotype of either deformed wing or lighter cuticle pigmentation/defects in cuticle sclerotization was observed for awd and th, respectively. However, for yel, no obvious phenotype was observed. Based on this, yel was selected as a target for the development of a CRISPR/Cas9 workflow to study gene knockout in E. heros. A total of 719 eggs were injected with the Cas9 nuclease (300 ng/mu L) together with the sgRNA (300 ng/mu L) targeting yel. A total of six insects successfully hatched from the injected eggs and one of the insects showed mutation in the target region, however, the phenotype was still not obvious. Overall, this study for the first time provides a useful CRISPR/Cas9 gene editing methodology to complement RNAi for functional genomic studies in one of the most important and economically relevant stink bug species