CRISPR/Cas9-mediated ebony knockout causes melanin pigmentation and prevents moth Eclosion in Ectropis grisescens.

Ectropis grisescens (Lepidoptera: Geometridae) is a destructive tea pest in China. Mimesis, characterized by changing body color, is an important trait of E. grisescens larvae. Hence, identifying melanin pathway-related genes may contribute to developing new pest control strategies. In the present study, we cloned Egebony, a gene potentially involved in melanin pigmentation in E. grisescens, and subsequently conducted CRISPR/Cas9-mediated targeted mutagenesis of Egebony to analyze its role in pigmentation and development. At the larvae, prepupae, and pupae stages, Egebony-knockout individuals exhibited darker pigmentation than the wild-type. However, Egebony knockout did not impact the colors of sclerotized appendants, including ocelli, setae, and claws. While mutant pupae could successfully develop into moths, they were unable to emerge from the puparium. Notably, embryo hatchability and larval survival of mutants remained normal. Further investigation indicated that mutant pupae exhibited significantly stronger shearing force than the wild-type, with the pigmented layer of mutant pupae appearing darker and thicker. Collectively, these results suggest that the loss of Egebony might increase the rigidity of the puparium and prevent moth eclosion. This study provides new insights into understanding the function and diversification of ebony in insect development and identifies a lethal gene that can be manipulated for developing effective pest control strategies.

Olfactory Gene Families in Scopula subpunctaria and Candidates for Type-II Sex Pheromone Detection.

Scopula subpunctaria, an abundant pest in tea gardens, produce type-II sex pheromone components, which are critical for its communicative and reproductive abilities; however, genes encoding the proteins involved in the detection of type-II sex pheromone components have rarely been documented in moths. In the present study, we sequenced the transcriptomes of the male and female S. subpunctaria antennae. A total of 150 candidate olfaction genes, comprising 58 odorant receptors (SsubORs), 26 ionotropic receptors (SsubIRs), 24 chemosensory proteins (SsubCSPs), 40 odorant-binding proteins (SsubOBPs), and 2 sensory neuron membrane proteins (SsubSNMPs) were identified in S. subpunctaria. Phylogenetic analysis, qPCR, and mRNA abundance analysis results suggested that SsubOR46 may be the Orco (non-traditional odorant receptor, a subfamily of ORs) of S. subpunctaria. SsubOR9, SsubOR53, and SsubOR55 belonged to the pheromone receptor (PR) clades which have a higher expression in male antennae. Interestingly, SsubOR44 was uniquely expressed in the antennae, with a higher expression in males than in females. SsubOBP25, SsubOBP27, and SsubOBP28 were clustered into the moth pheromone-binding protein (PBP) sub-family, and they were uniquely expressed in the antennae, with a higher expression in males than in females. SsubOBP19, a member of the GOBP2 group, was the most abundant OBP in the antennae. These findings indicate that these olfactory genes, comprising five candidate PRs, three candidate PBPs, and one candidate GOBP2, may be involved in type II sex pheromone detection. As well as these genes, most of the remaining SsubORs, and all of the SsubIRs, showed a considerably higher expression in the female antennae than in the male antennae. Many of these, including SsubOR40, SsubOR42, SsubOR43, and SsubIR26, were more abundant in female antennae. These olfactory and ionotropic receptors may be related to the detection of host plant volatiles. The results of this present study provide a basis for exploring the olfaction mechanisms in S. subpunctaria, with a focus on the genes involved in type II sex pheromones. The evolutionary analyses in our study provide new insights into the differentiation and evolution of lepidopteran PRs.