CRISPR/Cas9‐mediated methoprene‐tolerant 1 knockout results in precocious metamorphosis of beet armyworm (Spodoptera exigua) only at the late larval stage

Abstract

Juvenile hormone (JH) controls almost every aspect of an insect, especially metamorphosis. Since RNA interference works on transcripts and is often insufficient in Lepidoptera, how JH affects larval development in these insects is not well studied. Using the CRISPR/Cas9 technique, we knocked out Spodoptera exigua methoprene‐tolerant 1 (SeMet1) gene of beet armyworm by modifying two sites in the coding region. However, SeMet1 knockout did not affect egg hatch rate or larval development at L1–L3 stages. In contrast to the consistent five larval instars of the control group, L4 SeMet1 mutants began to show signs of precocious metamorphosis, that is, small patches of pupal cuticle. Most L4 and all L5 SeMet1 mutants died for failing to shed their mosaic cuticles. RNA‐seq indicated that most genes encoding pupal cuticle proteins and chitinase genes were altered in SeMet1 mutant L4 larvae. SeKr‐h1, a key transcription factor in JH action was significantly down‐regulated in L3‐L5 larvae, while SeBR‐C, a pupal indicator was only upregulated in L4‐L5 larvae. These results suggested that S. exigua larvae may initially develop independently of JH, and involve SeMet1 in transducing JH signalling, leading to controlled larval metamorphosis at the late larval stage. We believe our findings will enhance better understanding of JH regulation of larval development.

SeBLOS2 knockout via CRISPR/Cas9 leads to the loss of larval integument coloration in Spodoptera exigua (Lepidoptera: Noctuidae)

“…In the G0 generation, the CRISPR/Cas9-mediated chimeric mutation frequency of SeBLOS2 was 70%-74%, similar to that of BmBLOS2 in the silkworm (51%-95%) and SLBLOS2 in S. exigua (62%-71%) (Wang et al, 2013;Zhu et al, 2017). Additionally, a high chimeric frequency for SeMet1 (33%-34%) was previously obtained in S. exigua using a similar procedure (Zhao et al, 2022). Chimeric frequencies induced by CRISPR/Cas9 can be influenced by multiple variables, such as Cas9 protein/sgRNA injection concentration, differences in gene structure, and the target sites.…”

“…RNA was extracted from larvae (first and fifth instar), prepupae, pupae (Day‐3), and adults (Day 1–3) by Trizol, and cDNA was synthesized by PrimeScript TM RT Master Mix (Takala) following the manufacturer's instructions. The housekeeping gene β‐actin was used as an endogenous reference for qPCR (Zhao et al, 2022). Specific primers for each gene ( SeBLOS2 and β ‐ actin ) were created using DNAMAN software (Table 1).…”

Toxicological, biological, and biochemical impacts of the egyptian lavender (Lavandula multifida L.) essential oil on two lepidopteran pests

“…This family has several important moth pests such as the cotton leafworm (Spodoptera littoralis) (Boisd) (Khalifa et al 2023), black cutworm (Agrotis ipsilon), (Hufnagel), fall armyworm, S. frugiperda (J.E. Smith) (Palli et al 2023), beet armyworm, S. exigua (Hübner) (Zhao et al 2023), cotton bollworm, Helicoverpa armigera (Hübner), corn earworm, H. zea (Boddie), European corn borer, Ostrinia nubilalis (Hübner), and cabbage moth, Mamestra brassicae (Linnaeus, 1758). As generalist herbivores, these noctuid pest moths are known to damage a variety of important agricultural crops including cotton, maize, soybean, vegetables, and others, leading to global losses to crop production estimated in the billions annually through yield reductions and management costs (Le Goff and Nauen 2021).…”

RNA Interference-Mediated Knockdown of Tryptophan 2,3-Dioxygenase and Kynurenine-3-Monooxygenase in Monochamus Alternatus: Implications for Insect Control

“…After innovation of zinc finger nucleases (ZFNs), upon emergence of transcription activator-like effecter nuclease (TALENs) and CRISPR/Cas, targeted genome-editing technology is becoming more efficient, less costly and easier to operate. In addition to common model insects such as D. melanogaster, Culex and Tribolium castaneum, the most updated targeted genome-editing technology, the CRISPR/Cas system, is also widely used in Diptera [9,10], Hymenoptera [11], Hemiptera [12], Lepidoptera [13][14][15] and Orthoptera [16]. Therefore, it is urgent to develop a feasible gene-editing system for M. alternatus and expand the system's prevention methods.…”