CRISPR/Cas9 mediated BLOS2 knockout resulting in disappearance of yellow strips and white spots on the larval integument in Spodoptera litura

Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific Sldsx and female-specific Sldsx isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.

Section: Discussionmentioning

confidence: 99%

Identification and functional characterization of doublesex gene in the testis of Spodoptera litura

Wen

Zheng

et al. 2018

“…Further, ZFN activity was first detected in a non-Drosophila insect. Subsequently, Takasu et al provided a nearly perfect marker gene, BmBLOS2 that controls the formation of uric acid granules in the larval epidermis; it can be used to verify the activity of other genome editing tools in B. mori and other Lepidoptera insects (Zhu et al, 2017).…”

Section: Development Of Genome Editing In B Morimentioning

confidence: 99%

Genome editing in Bombyx mori: New opportunities for silkworm functional genomics and the sericulture industry

Smagghe

Xia

2018

In recent years, research in life sciences has been remarkably revolutionized owing to the establishment, development and application of genome editing technologies. Genome editing has not only accelerated fundamental research but has also shown promising applications in agricultural breeding and therapy. In particular, the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology has become an indispensable tool in molecular biology owing to its high efficacy and simplicity. Genome editing tools have also been established in silkworm (Bombyx mori), a model organism of Lepidoptera insects with high economic importance. This has remarkably improved the level and scope of silkworm research and could reveal new mechanisms or targets in basic entomology and pest management studies. In this review, we summarize the progress and potential of genome editing in silkworm and its applications in functional genomic studies for generating novel genetic materials.

“…In Lepidoptera, CRISPR/Cas9 has been used to analyze gene function (Wang et al, 2013;Zhang et al, 2015;Liu et al, 2017;Xu et al, 2017;Zeng et al, 2017), to enhance antiviral responses , to increase silk production , and in pest control (Liu et al, 2016). In S. litura, the CRISPR/Cas9 system was used to knock out genes that function in embryo development (Bi et al, 2016), that encode a sex pheromone (Zhu et al, 2016), and that influence uric acid granule formation (Zhu et al, 2017). Although the CRISPR/Cas9 system functions in S. litura, there are difficulties in mutant identification.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9‐mediated ebony knockout results in puparium melanism in Spodoptera litura

Zhang

et al. 2019

Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) genome editing system to target the ebony gene in the non‐model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony‐specific guide RNAs into S. litura embryos, we successfully induced a typical ebony‐deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction‐based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.