RNAi and CRISPR/Cas9 as Functional Genomics Tools in the Neotropical Stink Bug, Euschistus heros

Abstract

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/µL) together with the sgRNA (300 ng/µL) 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.

Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar)

“…The absence of eye color in the G1, G2 and G3 individuals of the w line of GWSS is consistent with previous work documenting ommochromes in hemipteran eyes 12 , 16 , 19 , 20 , 26 . Furthermore, the red–orange eyes and ocelli of GWSS cn mutants are consistent with the discovery of pteridines in water strider and BPH eyes 20 , 28 and also indicate that the yellow and brown pigments seen in the wild-type eyes and ocelli do not result from pteridines but are most likely due to ommochrome pigments.…”

“…There was 100% transmission of the w alleles and G1, G2 and G3 individuals transheterozygous for two mutant w alleles were recovered. The w gene has been previously used in proof-of-concept experiments in five other hemipteran species: corn planthopper (CPH), neotropical stink bug, brown planthopper (BPH), milkweed bug, and whitefly 12 , 14 , 16 , 19 , 20 . The rates of CRISPR/Cas9 mutagenesis based on eye phenotypes of G0 insects varied significantly from 0.2–2.5% (whitefly), 0–27.3% (BPH), 32.4% (CPH), 33% (stinkbug) and 14.0–92.5% (milkweed bug).…”

“…Significant fitness costs of w mutations have been reported in other Hemiptera. For example, G1 insects with w mutant alleles could not be generated in CPH and stinkbug 12 , 16 . Similarly, transmission of w alleles to the G1 generation in BPH and whitefly was low (3% and 1%, respectively) 14 , 20 .…”

“…In contrast, CRISPR/Cas9-mediated knock-out mutagenesis in the Hemiptera was not reported until 2018. To date, this technology has been used in seven species within six families of the Hemiptera including: brown planthopper ( Nilaparvata lugens ), corn planthopper ( Peregrinus maidis ), milkweed bug ( Oncopeltus fasciatus) , linden bug ( Pyrrhocoris apterus) , neotropical stink bug ( Euschistus heros) , whitefly ( Bemisia tabaci) , and pea aphid ( Acyrthosiphon pisum) 12 – 21 .…”

Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives

“…Methods for efficient macromolecule delivery are essential to any CRISPR genome-editing system and the delivery strategy can be a substantial technological hurdle preventing deployment in target organisms. While temporally delayed by 3 to 5 years relative to the gene-editing advances in D. melanogaster , mosquitoes, the Lepidoptera, and the Coleoptera, CRISPR/Cas9-editing is now reported for nine species of Hemiptera with a total of 17 different genes being targeted ( Tables 2 – 4 ) ( Xue et al, 2018 ; Kotwica-Rolinska et al, 2019 ; Le Trionnaire et al, 2019 ; Zhao et al, 2019 ; Cagliari et al, 2020 ; Heu et al, 2020 ; Reding and Pick, 2020 ; Klobasa et al, 2021 ; Xue et al, 2021 ; Heu et al, 2022 ; Pacheco et al, 2022 ).…”