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.

Plant Volatiles Modulate Seasonal Dynamics between Hosts of the Polyphagous Mirid Bug Apolygus lucorum.

Plant-derived volatiles play a significant role in host selection of phytophagous insects, but their role in seasonal host shifts remain unclear. The polyphagous mirid bug Apolygus lucorum displays marked seasonal host alternation. During summer, volatiles from flowering plants play a key role in A. lucorum foraging. Though A. lucorum adults deposit overwintering eggs on jujube (Ziziphus jujuba) and grape (Vitis vinifera) during autumn, it is unclear whether plant volatiles equally mediate this host selection behavior. During 2015 and 2016, we found that population densities of A. lucorum adults on cotton (Gossypium hirsutum) during August were higher than those in September, whereas the opposite pattern was observed on fruit trees (i.e., jujube and grape). The dispersal factor of the adult population that dispersed from cotton fields during September was higher than in August, whereas opposite patterns were observed in the neighboring jujube/grape orchard. In Y-tube olfactometer trials, A. lucorum adults preferred cotton plant volatiles over fruit tree odors in August, whereas the opposite patterns were found in September. Three electro-physiologically active volatiles (butyl acrylate, butyl propionate and butyl butyrate) were identified from jujube and grape plants. During September, active volatiles are emitted in considerably greater amounts by jujube and grape than in August, while the amount of volatile emissions in cotton decreases in September. Temporal shifts in plant volatile emission thus may modulate host plant foraging of A. lucorum, and appear to guide its colonization of different host plants. Our findings help understand the role of plant volatiles in the host plant selection and seasonal dynamics of polyphagous herbivores.

Identification of cytochrome P450, odorant-binding protein, and chemosensory protein genes involved in Type II sex pheromone biosynthesis and transportation in the tea pest, Scopula subpunctaria.

Sex pheromone-based pest management technology has been widely used to monitor and control insect pests in the agricultural, forestry, and public health sectors. Scopula subpunctaria is a widespread tea pest in China with Type II sex pheromone components. However, limited information is available on the biosynthesis and transportation of Type II sex pheromone components. In this study, we constructed an S. subpunctaria sex pheromone gland (PG) transcriptome and obtained 85,246 transcripts. Cytochrome P450 monooxygenases (CYPs) thought to epoxidize dienes and trienes to epoxides in the PG and odorant-binding proteins (OBPs) and chemosensory genes (CSPs) thought to be responsible for the binding and transportation of sex pheromone components. In present study, a total of 79 CYPs, 29 OBPs and 17 CSPs were identified. We found that SsubCYP341A and SsubCYP341B_ortholog1 belonged to the CYP341 family and were more highly expressed in the PG than in the female body. Of these, SsubCYP341A was the seventh-most PG-enriched CYP in the PG transcriptome. Two CYP4 members, CYP340BD_ortholog2 and CYP4G, were the top two most PG-enriched CYPs. Tissue expression and phylogenetic tree analysis showed that SsubOBP25, 27, and 28 belonged to the moth pheromone-binding protein family; they were distinctly expressed in the antennae and were more abundant in male antennae than in female antennae. SsubCSP16 was distributed into the same clade as CSPs from other moths that showed high binding affinities to sex pheromone components. It indicated that all the above-mentioned genes could be involved in sex pheromone biosynthesis or transportation. Our study provides large-scale PG sequence information that can be used to identify potential targets for the biological control of S. subpunctaria by disrupting its sex pheromone biosynthesis and transportation pathways.

Perception of and Behavioral Responses to Host Plant Volatiles for Three Adelphocoris Species.

In China, the genus Adelphocoris (Hemiptera: Miridae) includes three dominant pest species (A. suturalis, A. lineolatus and A. fasciaticollis), which cause great damage to cotton, alfalfa and other crops. In this study, we examined the role of the major volatile organic compounds from plants in host location by these three insects. Gas chromatography-electroantennography and gas chromatography/mass spectrometry analyses identified seven electroantennogram (EAG)-active compounds from 11 host plants. Although the insects responded to all of these compounds in EAG trials, some compounds did not elicit behavioral responses in Y-tube olfactometer bioassays. Adelphocoris suturalis adults showed behavioral responses to four EAG-active compounds, n-butyl ether, butyl acrylate, butyl propionate and butyl butyrate. These four compounds, in addition to p-xylene, were also attractive to A. lineolatus adults. However, A. fasciaticollis adults were attracted only by butyl acrylate, butyl propionate and butyl butyrate. In field trials, A. suturalis and A. fasciaticollis were each attracted to five individual compounds (m-xylene, n-butyl ether, butyl acrylate, butyl butyrate and butyl propionate for A. suturalis and m-xylene, butyl acrylate, butyl butyrate, butyl propionate, and 1,8-cineole for A. fasciaticollis). By contrast, A. lineolatus aduts were attracted to six individual compounds, m-xylene, p-xylene, n-butyl ether, butyl acrylate, butyl butyrate, and butyl propionate. These compounds may be important in host plant location by the Adelphocoris species, and may be useful for developing attractants for adults of these species.

Mating and post-copulation behavior in the tea leafhopper, Empoasca onukii (Hemiptera: Cicadellidae).

The tea leafhopper, Empoasca onukii, relies on substrate-borne vibrations for sexual communication and is mainly controlled with chemical pesticides, which poses risks to the environment and food safety. Based on previous studies, we conducted a series of behavioral assays by simultaneous observation of vibration signals and movement to investigate the mating and post-copulation behavior of tea leafhoppers. During mating, the activity of E. onukii was restricted to dawn and dusk and concentrated on the sixth or seventh mature leaf below the tea bud. By comparing the time spent in locating females among different males, the timely reply of females was the key factor affecting mating success. Empoasca onukii females mated only once in their lives, while males could mate multiple times. Male rivalry behavior involved two distinct strategies. The rivals could send disruptive pulses to overlap the male calling signals, locate the courting males, and drive them away after contact. Some rivals could emit mating disruption signals (MDSs) to interrupt the ongoing identification duet and establish their own mating communication. Both identification and location duets could be interrupted by playback of MDSs, which is essential to create effective synthetic signals to disrupt mating communication of E. onukii. Our study clarified the spatial and temporal distribution of E. onukii in mating and the function of MDSs, which will be essential to develop future vibrational mating disruption techniques for E. onukii and its energy-efficient application in the field.