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.

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.

Measurement of volatile plant compounds in field ambient air by thermal desorption-gas chromatography-mass spectrometry.

Determination of volatile plant compounds in field ambient air is important to understand chemical communication between plants and insects and will aid the development of semiochemicals from plants for pest control. In this study, a thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) method was developed to measure ultra-trace levels of volatile plant compounds in field ambient air. The desorption parameters of TD, including sorbent tube material, tube desorption temperature, desorption time, and cold trap temperature, were selected and optimized. In GC-MS analysis, the selected ion monitoring mode was used for enhanced sensitivity and selectivity. This method was sufficiently sensitive to detect part-per-trillion levels of volatile plant compounds in field ambient air. Laboratory and field evaluation revealed that the method presented high precision and accuracy. Field studies indicated that the background odor of tea plantations contained some common volatile plant compounds, such as (Z)-3-hexenol, methyl salicylate, and (E)-ocimene, at concentrations ranging from 1 to 3400 ng m(-3). In addition, the background odor in summer was more abundant in quality and quantity than in autumn. Relative to previous methods, the TD-GC-MS method is more sensitive, permitting accurate qualitative and quantitative measurements of volatile plant compounds in field ambient air.

Identification and field evaluation of non-host volatiles disturbing host location by the tea geometrid, Ectropis obliqua.

Volatile organic compounds derived from non-host plants, Ocimum basilicum, Rosmarinus officinalis, Corymbia citriodora, and Ruta graveolens, can be used to mask host plant odors, and are repellent to the tea geometrid, Ectropis obliqua. Volatile compounds were collected by headspace absorption, and the components were identified and quantified by using gas chromatography/mass spectrometry. The responses of antennae of female E. obliqua to the compounds were evaluated with gas chromatography/electroantennography detection. Qualitative and quantitative differences were found among the four odor profiles. Consistent electroantennographic activity was obtained for eight of the volatiles from the four plants: ß-myrcene, α-terpinene, γ-terpinene, linalool, cis-verbenol, camphor, α-terpineol, and verbenone. In a Y-tube bioassay, six chemicals, ß-myrcene, γ-terpinene, (R)-(-)-linalool, (S)-(-)-cis-verbenol, (R)-(+)-camphor, and (S)-(-)-verbenone, were the main compounds responsible for repelling E. obliqua. An eight-component mixture including all of the bioactive compounds (in a ratio of 13:2:13:8:1:24:6:17) from R. officinalis was significantly more effective at repelling the moths than any single compound or a mixture of equal amounts of the eight compounds. Field results demonstrated that intercropping tea plants with R. officinalis effectively suppressed E. obliqua infestations in a tea plantation. Our findings suggests that odor blends of R. officinalis play a role in disturbing host orientation behavior, and in repelling E. obliqua adults, and that R. officinalis should be considered when developing "push-pull" strategies aimed at optimizing the control of E. obliqua with semiochemicals.

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.

Anatomical Comparison of Antennal Lobes in Two Sibling Ectropis Moths: Emphasis on the Macroglomerular Complex.

Ectropis obliqua and Ectropis grisescens are two sibling moth species of tea plantations in China. The male antennae of both species can detect shared and specific sex pheromone components. Thus, the primary olfactory center, i.e., the antennal lobe (AL), plays a vital role in distinguishing the sex pheromones. To provide evidence for the possible mechanism allowing this distinction, in this study, we compared the macroglomerular complex (MGC) of the AL between the males of the two species by immunostaining using presynaptic antibody and propidium iodide (PI) with antennal backfills, and confocal imaging and digital 3D-reconstruction. The results showed that MGC of both E. obliqua and E. grisescens contained five glomeruli at invariant positions between the species. However, the volumes of the anterior-lateral glomerulus (ALG) and posterior-ventral (PV) glomerulus differed between the species, possibly related to differences in sensing sex pheromone compounds and their ratios between E. obliqua and E. grisescens. Our results provide an important basis for the mechanism of mating isolation between these sibling moth species.

Foliage Intensity is an Important Cue of Habitat Location for Empoasca onukii.

For many herbivorous insects, vision is more important than olfaction in the prealighting stage of host habitat location. Tea leafhoppers, Empoasca onukii (Hemiptera, Cicadellidae), are serious pests that preferentially inhabit the tender leaves of tea plants across China. Here, we investigated whether tea leafhoppers could distinguish foliage colors associated with different leaf ages and use this visual cue to guide suitable habitat location from short distances. Similar to honeybees, the adult E. onukii has an apposition type of compound eye, and each ommatidium has eight retinular cells, in which three spectral types of photoreceptors are distributed, with peak sensitivities at 356 nm (ultraviolet), 435 nm (blue), and 542 nm (green). Both changes in spectral intensity and hue of reflectance light of the host foliage were correlated with varying leaf age, and the intensity linearly decreased with increasing leaf age. Behavioral responses also showed that adult E. onukii could discriminate between the simulated colors of host foliage at different leaf ages without olfactory stimuli and selected the bright colors that strongly corresponded to those of tender leaves. The results suggest that, compared with the spectral composition (hue), the intensity of light reflectance from leaves at different ages is more important for adult leafhoppers when discriminating host foliage and could guide them to tender leaves at the top of tea shoots.

Geographical Distribution of Ectropis grisescens (Lepidoptera: Geometridae) and Ectropis obliqua in China and Description of an Efficient Identification Method.

Ectropis grisescens Warren 1894 (Lepidopotera: Geometridae) and Ectropis obliqua Prout 1915 (Lepidopotera: Geometridae) are the most destructive chewing pests in China's tea plantations. Ectropis grisescens sex pheromone lures and E. obliqua nucleopolyhedrosis virus (EoNPV) are two species-specific and effective bio-control technologies to control these pests. Because these two species are morphologically similar, tea growers are unable to discriminate them by visual inspection. Hence, determining whether to use E. grisescens sex pheromone lures or EoNPV is difficult without knowledge on the geographical distribution of these two Ectropis species in China. In this study, we developed an efficient identification method, which is considerably cheaper and faster than sequencing the cytochrome c oxidase I gene. Overall, 2,588 E. grisescens and E. obliqua samples, collected from 13 provinces and municipalities in China covering the major regions where these pests have been reported, were identified. All insect samples from southern Jiangsu Province were identified as E. obliqua. Both Ectropis species were mix-distributed at the Anhui-Zhejiang Province border areas, whereas E. grisescens was mostly collected from the other sampling sites. Thus, E. obliqua might be mainly distributed at the junction of Jiangsu, Anhui, and Zhejiang Provinces. In contrast, E. grisescens has a considerably wide distribution area and is the main lepidopteran pest in the tea plantations of China. Our results contribute to improve the management of E. grisescens and E. obliqua populations and provide new insights for further studies on these two species.

[Jasmonates: From induced plant anti-herbivore defensive reaction to growth-defense tradeoffs]. / èŒ‰èŽ‰é ¸ç±»åŒ–åˆç‰©:从植物的诱导抗虫防御反应到生长-防御权衡.

Jasmonates (JAs), which can exogenously induce anti-herbivore defensive reaction in plants, are important in the field of plant physiology and plant protection. We summarized the ecological effects of exogenous JAs applied on 40 species of plants on various herbivores (such as Lepidoptera and Hemipteran) and their natural enemies in the past 20 years. We systematically genera-lized the research status about the direct and indirect antiherbivore defense induced by JAs, induced systemic defense, induction methods, induced performance in field, and the application status of JAs. Moreover, combining with the latest literatures, we reviewed the effects of JAs on plant growth-defense trade-offs from the cross-talk between phytohormone signaling pathways, and the regulation of nodes in the JA signaling pathway. Finally, we proposed the future directions and key aspects of the research on the plant anti-herbivore defense induced by exogenous JAs, which would promote the development of the related research and the application of JAs in field.

Design of an Attractant for Empoasca onukii (Hemiptera: Cicadellidae) Based on the Volatile Components of Fresh Tea Leaves.

The tea leafhopper, Empoasca onukii Matsuda, is a serious pest of the tea plant. E. onukii prefers to inhabit vigorously growing tender tea leaves. The host selection of E. onukii adults may be associated with plant volatile compounds (VOCs). We sought to identify potentially attractive VOCs from tea leaves at three different ages and test the behavioral responses of E. onukii adults to synthetic VOC blends in the laboratory and field to aid in developing an E. onukii adult attractant. In darkness, the fresh or mature tea leaves of less than 1-mo old could attract more leafhoppers than the mature branches (MB) that had many older leaves (leaf age >1 mo). Volatile analysis showed that the VOC composition of the fresh leaves was the same as that of the mature leaves, but linalool and indole were not at detectable levels in VOCs from the MB. Moreover, the mass ratio differed for each common volatile in the three types of tea leaves. When under competition with volatiles from the MB, the leafhoppers showed no significant tropism to each single volatile but could be attracted by the synthetic volatile blend imitating the fresh leaves. With the removal of some volatile components, the effective synthetic volatile blend was mixed with (Z)-3-hexen-1-ol, (Z)-3-hexenyl acetate, and linalool at a mass ratio of 0.6:23:12.6. These three volatiles may be the key components for the host selection of E. onukii adults and could be used as an attractant in tea gardens.

Comparison of Olfactory Genes in Two Ectropis Species: Emphasis on Candidates Involved in the Detection of Type-II Sex Pheromones.

The sibling species Ectropis grisescens and E. obliqua are the major chewing tea pests in China. A difference in sex pheromone components plays a central role in premating isolation in these two species. To investigate the mechanism of premating isolation in these two Ectropis species, we sequenced the transcriptomes of the antennae of female and male E. obliqua individuals and performed phylogenetic analyses, abundance analyses, and tissue expression profile analyses to compare the olfactory genes involved in the detection of sex pheromones. A total of 36 odorant-binding proteins (OBPs) and 52 olfactory receptors (ORs) were identified in E. obliqua. Phylogenetic analyses showed that EoblOBP2, 3, and 25 were grouped in the pheromone-binding protein clade with EgriOBP2, 3, 25, and another lepidopteran PBP. EoblOR25 and 28 were grouped with EgriOR25, 28, and pheromone receptors for the detection of Type-I sex pheromone components. EoblOR24, 31, 37, and 44 were grouped with EgriOR24, 31, 37, and 44. All of these 4 EoblORs and 4 EgriORs showed higher abundance in male antennae than in female ones. Therefore, OBP2, 3, 25 and OR24, 31, 37, 44 of E. grisescens and E. obliqua might be responsible for sex pheromone component detection. However, the sequences of these genes in E. grisescens and E. obliqua were more than 90% identical. This indicates that these orthologous genes might play similar roles in the detection of sex pheromones. In contrast, the observed OBPs and ORs differed in abundance between the antennae of the two Ectropis species. Therefore, we speculate that these two Ectropis species use the different transcript levels of PRs to differentiate sex pheromone components. The results of the present study might contribute in deciphering the mechanism for premating isolation in these species and may be of use in devising strategies for their management.

Gene Identification of Pheromone Gland Genes Involved in Type II Sex Pheromone Biosynthesis and Transportation in Female Tea Pest Ectropis grisescens.

Moths can biosynthesize sex pheromones in the female sex pheromone glands (PGs) and can distinguish species-specific sex pheromones using their antennae. However, the biosynthesis and transportation mechanism for Type II sex pheromone components has rarely been documented in moths. In this study, we constructed a massive PG transcriptome database (14.72 Gb) from a moth species, Ectropis grisescens, which uses type II sex pheromones and is a major tea pest in China. We further identified putative sex pheromone biosynthesis and transportation-related unigenes: 111 cytochrome P450 monooxygenases (CYPs), 25 odorant-binding proteins (OBPs), and 20 chemosensory proteins (CSPs). Tissue expression and phylogenetic tree analyses showed that one CYP (EgriCYP341-fragment3), one OBP (EgriOBP4), and one CSP (EgriCSP10) gene displayed an enriched expression in the PGs, and that EgriOBP2, 3, and 25 are clustered in the moth pheromone-binding protein clade. We considered these our candidate genes. Our results yielded large-scale PG sequence information for further functional studies.

Chemosensory Gene Families in Ectropis grisescens and Candidates for Detection of Type-II Sex Pheromones.

Tea grey geometrid (Ectropis grisescens), a devastating chewing pest in tea plantations throughout China, produces Type-II pheromone components. Little is known about the genes encoding proteins involved in the perception of Type-II sex pheromone components. To investigate the olfaction genes involved in E. grisescens sex pheromones and plant volatiles perception, we sequenced female and male antennae transcriptomes of E. grisescens. After assembly and annotation, we identified 153 candidate chemoreception genes in E. grisescens, including 40 odorant-binding proteins (OBPs), 30 chemosensory proteins (CSPs), 59 odorant receptors (ORs), and 24 ionotropic receptors (IRs). The results of phylogenetic, qPCR, and mRNA abundance analyses suggested that three candidate pheromone-binding proteins (EgriOBP2, 3, and 25), two candidate general odorant-binding proteins (EgriOBP1 and 29), six pheromone receptors (EgriOR24, 25, 28, 31, 37, and 44), and EgriCSP8 may be involved in the detection of Type-II sex pheromone components. Functional investigation by heterologous expression in Xenopus oocytes revealed that EgriOR31 was robustly tuned to the E. grisescens sex pheromone component (Z,Z,Z)-3,6,9-octadecatriene and weakly to the other sex pheromone component (Z,Z)-3,9-6,7-epoxyoctadecadiene. Our results represent a systematic functional analysis of the molecular mechanism of olfaction perception in E. grisescens with an emphasis on gene encoding proteins involved in perception of Type-II sex pheromones, and provide information that will be relevant to other Lepidoptera species.

Identification and Comparative Study of Chemosensory Genes Related to Host Selection by Legs Transcriptome Analysis in the Tea Geometrid Ectropis obliqua.

Host selection by female moths is fundamental to the survival of their larvae. Detecting and perceiving the non-volatile chemicals of the plant surface involved in gustatory detection determine the host preference. In many lepidopteran species, tarsal chemosensilla are sensitive to non-volatile chemicals and responsible for taste detection. The tea geometrid Ectropis obliqua is one devastating chewing pest selectively feeding on limited plants, requiring the specialized sensors to forage certain host for oviposition. In present study, we revealed the distribution of chemosensilla in the ventral side of female fifth tarsomere in E. obliqua. To investigate its molecular mechanism of gustatory perception, we performed HiSeq 2500 sequencing of the male- and female- legs transcriptome and identified 24 candidate odorant binding proteins (OBPs), 21 chemosensory proteins (CSPs), 2 sensory neuron membrane proteins (SNMPs), 3 gustatory receptors (GRs) and 4 odorant receptors (ORs). Several leg-specific or enriched chemosensory genes were screened by tissue expression analysis, and clustered with functionally validated genes from other moths, suggesting the potential involvement in taste sensation or other physiological processes. The RPKM value analysis revealed that 9 EoblOBPs showed sex discrepancy in the leg expression, 8 being up-regulated in female and only 1 being over expressed in male. These female-biased EoblOBPs indicated an ecological adaption related with host-seeking and oviposition behaviors. Our work will provide basic knowledge for further studies on the molecular mechanism of gustatory perception, and enlighten a host-selection-based control strategy of insect pests.