Identification of a unique three-component sex pheromone produced by the tea black tussock moth, Dasychira baibarana (Lepidoptera: Erebidae: Lymantriinae).

BACKGROUND: The tea black tussock moth Dasychira baibarana Matsumura is a devastating pest in tea plantations that causes substantial economic losses. Presently, there is no effective method to control this pest other than pesticide application. The identified sex pheromone of D. baibarana could be used for detecting and monitoring this pest. RESULTS: Gas chromatography-electroantennogram detection showed that D. baibarana male moth antennae responded strongly to three components in the female sex pheromone gland, which were identified as: (3Z,6Z)-cis-9,10-epoxyhenicosa-3,6-diene (Z3,Z6,epo9-21:H) (I), (3Z,6Z,11E)-cis-9,10-epoxyhenicosa-3,6,11-triene (Z3,Z6,epo9,E11-21:H) (II) and (3Z,6Z)-henicosa-3,6-dien-11-one (Z3,Z6-21:11-one) (III). Dasychira baibarana uses a unique composition of an epoxydiene, epoxytriene, and dienone with the same 3,6-dienyl motif as its sex pheromone. The epoxytriene and dienone were not previously characterized as insect pheromone components. Electroantennogram analysis showed that each synthetic compound strongly stimulated male antennae, and compounds II and III elicited stronger responses than compound I. A wind tunnel bioassay and field trapping experiments proved that, ternary blends of compounds I-III attracted D. baibarana. Efficient attraction was achieved with a rubber septum baited with 500 µg of a mixture of compounds I-III at the ratio 25:20:55. CONCLUSION: The three identified compounds elicited an electroantennogram response in D. baibarana male moth antennae, and a mixture of the three components at the ratio 25:20:55 attracted D. baibarana male moths in a wind tunnel assay and field trapping experiments. This blend could be used for integrated management of D. baibarana in tea plantations. © 2022 Society of Chemical Industry.

Electrophysiological and Behavioral Responses of Dasychira baibarana (Lepidoptera: Lymantriidae) to Tea Plant Volatiles.

Tea black tussock moth, Dasychira baibarana (Matsumura) (Lepidoptera: Lymantriidae), is a devastating pest species of the tea plant in China. Here, we evaluated the responses of D. baibarana to tea plant volatiles using gas chromatography coupled electroantennographic detection (GC-EAD), eleclectroantennography (EAG), and a Y-tube olfactometer. In total, 11 of 18 analyzed compounds elicited GC-EAD responses from test insects. GC-EAD bio-active compounds were further investigated using EAG and behavioral responses. In the EAG analysis, male moths had significantly greater responses to four compounds [(Z)-3-hexenyl butyrate, (Z)-3-hexen-1-ol, ocimene and benzyl alcohol] than female moths. For females, maximum EAG amplitudes, were recorded in response to linalool, (Z)-3-hexenyl hexanoate and (Z)-jasmone. In EAG and behavioral bio-assays, the responses of both sexes were dose independent. In behavioral bio-assays male moths responding significantly to (Z)-3-hexen-1-ol, ocimene, (Z)-3-hexenyl butyrate, linalool, benzyl alcohol, and (Z)-jasmone at various concentrations. For females, significant behavioral responses were observed to (Z)-3-hexenyl hexanoate, followed by (Z)-jasmone, linalool, ocimene, and benzyl alcohol. However, neither sex was sensitive to 4 of the 11 tested compounds, phenyethyl alcohol, phenylacetonitrile, (E)-nerolidol, and indole. The present results showed that tea plant volatiles influenced the behavior of D. baibarana moths, which will greatly contribute in developing eco-friendly control strategies for D. baibarana, through the application of a blend of compounds that showed significant EAG and behavioral responses or a blend combined with female-produced sex pheromones.

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.

Comparative transcriptomic analysis of resistant and susceptible tea cultivars in response to Empoasca onukii (Matsuda) damage.

MAIN CONCLUSION: Transcriptomic studies in resistant and susceptible tea cultivars have been performed to reveal the different defense molecular mechanisms of tea after E. onukii feeding. The molecular mechanism by which tea plants respond to small green leafhopper Empoasca onukii (Matsuda) damage is unclear. Using the resistant tea plant cultivar Juyan (JY) and the susceptible tea plant cultivar Enbiao (EB) as materials, this study performed RNA-seq on tea leaf samples collected at three time points (6 h, 12 h, 24 h) during exposure of the plants to leafhopper to reveal the molecular mechanisms that are activated in susceptible and resistant tea plant cultivars in response to leafhopper damage. The numbers of DEGs in the susceptible tea cultivar during early (6 h) and late (24 h) stages of leafhopper induction were higher than those in the resistant cultivar at the same time points. The stress responses to leafhopper were most intense at 12 h in both tea cultivars. Pathway enrichment analysis showed that most up-regulated DEGs and their related metabolic pathways were similar in the two tea cultivars. However, during the early stage of leafhopper induction (6 h), jasmonic acid (JA)-related genes were significantly up-regulated in the resistant cultivar. The terpenoid biosynthetic pathway and the α-linolenic acid metabolic pathway were activated earlier in the resistant cultivar and remained activated until the late stage of leafhopper damage. Our results confirmed that after leafhopper damage, the resistant tea cultivar activated its defense responses earlier than the susceptible cultivar, and these defense responses were mainly related to terpenoid metabolism and JA biosynthetic pathway. The results provide important clues for further studies on resistance strategy of tea plants to pest.

Primary screening and application of repellent plant volatiles to control tea leafhopper, Empoasca onukii Matsuda.

BACKGROUND: The tea leafhopper, Empoasca onukii Matsuda (Hemiptera: Cicadellidae), is a major pest of tea plants in China. Here, we evaluated the repellent properties of eight volatile chemicals alone and in various combinations as tools for the management of this pest in tea gardens. These chemicals were from the Alliaceae and other aromatic plants, and are known to repel various insect species. RESULTS: Among the eight volatile compounds, dimethyl disulfide (DMDS), 1,8-cineole and allyl methyl sulfide were significantly repellent towards E. onukii adults. DMDS and 1,8-cineole were mixed to formulate a binary repellent. Under field conditions, spraying and slow-release applications of the mixture significantly decreased the density of E. onukii adults. The repelling effect after spraying was very short, only ∼ 2 days, but the slow-release mixture had a longer term repelling effect on E. onukii adults. High emission of the slow-release mixture, which was achieved by increasing the number of slow-release bottles, had a stronger repellent effect than low emission. Moreover, when the amount emitted was sufficient, the slow-release mixture significantly decreased the number of leafhopper nymphs in a treated tea-plant line, and significantly decreased the number of leafhopper adults and nymphs in a tea-plant line adjacent to the treated area. CONCLUSION: This study demonstrates the repellent action of a mixture of DMDS and 1,8-cineole applied by a slow-release method against E. onukii in a tea plantation. This mixture has potential applications in integrated pest management schemes. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A Disease Resistance Elicitor Laminarin Enhances Tea Defense against a Piercing Herbivore Empoasca (Matsumurasca) onukii Matsuda.

The tea plant (Camellia sinensis) suffers heavily from a harmful piercing pest, the tea green leafhopper (TLH) Empoasca (Matsumurasca) onukii Matsuda. In the present study, we studied the effect of an efficient elicitor of plant disease resistance, the ß-1,3-glucan laminarin, on the induced defense against TLH in tea plants. Defense responses elicited by laminarin in tea include the activation of mitogen-activated protein kinases and WRKY, the burst of H2O2, salicylic acid, and abscisic acid, and the accumulation of direct-defense chemicals (including chitinase, phenylalanine ammonia lyase, callose, polyphenol oxidase, and flavonol synthase), as well as the production of volatile compounds. The laminarin-treated tea plants reduced the performance of TLH and enhanced the attractiveness to the egg parasitoid wasp of TLH, Stethynium empoascae Subba Rao. In the field experiment, laminarin application effectively reduced the number of TLH by attracting parasitoids. These results suggest that laminarin can induce protection against TLH by regulating signaling pathways in tea plant. Our study also proposes an environment friendly strategy for the integrated management of an economically important piercing pest.

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.

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.

Does Background Odor in Tea Gardens Mask Attractants? Screening and Application of Attractants for Empoasca onukii Matsuda.

Plant volatiles help herbivores to locate their hosts, and therefore, they could be used to help develop pesticide-free pest management strategies. To develop an attractant for tea leafhopper (Empoasca onukii), we screened nine tea plant volatile compounds for their attractiveness using Y-tube olfactometer assays. Results indicated that tea leafhoppers significantly preferred ocimene, limonene, (Z)-3-hexenol, and (Z)-3-hexenyl acetate over clean air. These compounds were combined in a blend which lost its attractiveness at concentrations below 10-2 g/ml in liquid paraffin. In field tests, the blend was attractive to leafhoppers only in autumn, but not in summer. Analyses of the tea field background odor showed that all four components of the blend were present at much higher concentrations in summer (0.05-0.001 ng/liter) than in autumn (~10- to 25-fold lower). In field Y-tube bioassays, compared with the tea field background odor, the blend was attractive at a concentration of 10-1 g/ml in liquid paraffin, but not at 10-2 g/ml. These results suggest that field background odor can disrupt the attractiveness of an attractant based on plant volatiles to herbivores.

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.

TTF1-induced apoptosis of HepG-2 cells through a mitochondrial pathway.

The Chinese medicinal herb Sorbaria sorbifolia, native to Changbai Mountain, can induce apoptosis in HepG-2 cells. We studied the mechanism by which 5, 2', 4'-trihydroxy-6,7, 5'-trimethoxyflavone (TTF1) isolated from acetic ether extracts of Sorbaria sorbifolia induces apoptosis in HepG-2 cells. The results showed that TTF1 both inhibited cell growth and induced apoptosis. RT-PCR and Western blot analysis showed that TTF1 treatment led to decreased transcription and protein expression of bcl-2 and an increase in bax, Cyt-c, caspase-3 and caspase-9. These results together suggest that TTF1 may induce apoptosis of HepG-2 cells through a mitochondrial pathway.