Genome editing in the fall armyworm, Spodoptera frugiperda: Multiple sgRNA/Cas9 method for identification of knockouts in one generation

Zhu, Guan-Heng; Chereddy, Shankar C. R. R.; Howell, Jeffrey L.; Palli, Subba Reddy

doi:10.1016/j.ibmb.2020.103373

Cited by 48 publications

(22 citation statements)

References 48 publications

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“…To do this, we used CRISPR NHEJ targeting the gene Biogenesis of lysosome-related organelles complex one subunit 2 ( Blos2 ). Blos2 is located on the lepidopteran Z sex chromosome and mediates the formation of pigment-containing organelles, with viable knockout alleles generating translucent larval epidermis in several moth species ( Cheli et al., 2010 ; Fujii et al., 2010 ; Lee et al., 2018 ; Nakade et al., 2014 ; Zhu et al., 2020 ). CRISPR mutagenesis induced G 0 mosaic phenotypes in the larval integument, with mutant portions lacking the characteristic pink pigmentation observed in WT individuals ( Figure S3 A).…”

Section: Resultsmentioning

confidence: 99%

Mapping and CRISPR homology-directed repair of a recessive white eye mutation in Plodia moths

et al. 2022

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Section: Resultsmentioning

confidence: 99%

Mapping and CRISPR homology-directed repair of a recessive white eye mutation in Plodia moths

et al. 2022

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“…To generate transgenic animals, a solution containing piggyBac:hr5ie1-EGFP:ie1-CeSid1 plasmid (300 ng/μL) and HyPBT mRNA (200 ng/μL) was injected into fresh S. frugiperda eggs (less than 4 hr old) as previously described [26]. Injected eggs were kept at 27 ± 1°C, 60 ± 10% RH until hatching.…”

Section: S Frugiperdamentioning

confidence: 99%

Caenorhabditis eleganssystemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner

et al. 2020

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RNA interference (RNAi) is an important tool for gene function studies in insects, especially in non-model insects. This technology is also being developed for pest control. However, variable RNAi efficiency among insects is limiting its use in insects. Systemic RNAi in Caenorhabditis elegans requires systemic RNA interference defective protein 1 (CeSid1). The expression of CeSid1 in insect cell lines was shown to improve RNAi. However, the mechanisms through which this double-stranded RNA (dsRNA) transporter improves RNAi efficiency in insects is not known. We stably expressed CeSid1 in two Spodoptera frugiperda cell lines, Sf9 and Sf17 cells derived from ovary and midgut, respectively. Expression of CeSid1 enhanced RNAi efficiency in ovarian Sf9 cells, but not in midgut Sf17 cells. Reduced accumulation of dsRNA in late endosomes and successful processing dsRNA to siRNA contribute to enhanced RNAi efficiency in Sf9 cells. Transgenic S. frugiperda expressing CeSid1 were produced and tested for RNAi efficiency. RNAi efficiency enhancement due to CeSid1 expression showed tissue specificity. Compared to RNAi efficiency in wild-type S. frugiperda, CeSid1 expressing transgenic S. frugiperda showed a significant improvement of RNAi in tissues such as Verson's glands. In contrast, no improvement in RNAi was observed in tissues such as midgut. The in vitro cell-type specific and in vivo tissue-specific enhancement of RNAi efficiency by CeSid1 in S. frugiperda provides valuable information for improving RNAi in insects such as those belonging to order Lepidoptera where RNAi is variable and inefficient.

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“…In order to somatically disrupt the Oatp74D gene in vivo , CRISPR-Cas9 was performed by injecting S. frugiperda eggs according to a previously established protocol [44]. Briefly, egg batches were collected shortly after the onset of the scotoperiod and transferred to double sided tape using a whetted paintbrush [26].…”

Section: Methodsmentioning

confidence: 99%

Functional characterization of putative ecdysone transporters in lepidopteran pests

Samantsidis

Fotiadou

Tzavellas

et al. 2022

Preprint

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The insect steroid hormone ecdysone plays a critical role in insect development. Several recent studies have shown that ecdysone is transported through Organic Anion Transporting Polypeptides (OATPs) in insects such as flies and mosquitoes. However, the conservation of this mechanism across other arthropods and the role of this transporter in canonical ecdysone pathways are less well studied. Herein we functionally characterized the putative ecdysone transporter OATP74D from two major agricultural moth pests: Helicoverpa armigera (cotton bollworm) and Spodoptera frugiperda (fall armyworm). Phylogenetic analysis of OATP transporters across the superphylum Ecdysozoa revealed that Oatp74D is well represented among arthropod species and appeared only at the root of the arthropod lineage. Partial disruption of Oatp74D in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential for development in vivo. Depletion and re-expression of OatP74D in the lepidoptera cell line RP-HzGUT-AW1(MG) confirmed the gene’s role in ecdysone import and demonstrated that OATP74D is essential for the transcriptional activation of ecdysone responsive genes including caspase-3, implicating this transporter in cell death pathways. Establishment of a simple and robust luciferase assay using the RP-HzGUT- AW1(MG) cell line demonstrated that both HaOATP74D and SfOATP74D are inhibited by rifampicin, a well-known organic anion transporter inhibitor. Overall, this work sheds more light on ecdysone uptake mechanisms across insect species and broadens our knowledge of the physiological roles of OATPs in the transportation of endogenous substrates.Author SummaryThe insect steroid hormone ecdysone is critical in regulating many aspects of insects’ life, including development and reproduction. A passive diffusion model was never functionally resolved, but was strongly supported until an organic anion transporting polypeptide was identified to mediate the transport of the hormone. The OATP74D, belonging to the Solute carrier superfamily, has been identified and functionally characterized for the first time in Drosophila melanogaster. Although phylogenetic analysis suggests that the Drosophila Oatp74D is probably conserved among several insect species, the theory for transporter mediated ecdysone uptake cannot be generalized to all insects without concrete proof. In here we provide functional evidence that the Oatp74D of two lepidopteran pest species: Helicoverpa armigera and Spodoptera frugiperda, is highly required for insect survival and development. Furthermore, we reveal that the OATP74D is necessary to regulate the expression of several ecdysone response genes, including caspase-3 which is involved in programmed cell death. In addition, we have developed a cell-based platform for screening chemical compounds against the lepidopteran orthologs of Oat74D and rifampicin was functionally shown to inhibit ecdysone uptake. Taken all together, our study reveals that Oatp74D is conserved among several arthropod species in the ecdysone pathway and given the high necessity for an effective control of these two lepidopteran species, we hypothesized that OATP74D could serve as a possible drug target in those two species.

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Genome editing in the fall armyworm, Spodoptera frugiperda: Multiple sgRNA/Cas9 method for identification of knockouts in one generation

Cited by 48 publications

References 48 publications

Mapping and CRISPR homology-directed repair of a recessive white eye mutation in Plodia moths

Mapping and CRISPR homology-directed repair of a recessive white eye mutation in Plodia moths

Caenorhabditis eleganssystemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner

Functional characterization of putative ecdysone transporters in lepidopteran pests

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