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.

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.

Tea and human health: biomedical functions of tea active components and current issues.

Originating in China, tea and tea planting have spread throughout the world since the middle of the Tang dynasty. Now people from 160 countries in the world are accustomed to tea drinking. A brief history of tea's medicinal role in China and its spread to the world are introduced. The effectiveness of tea active components and tea drinking on major human diseases, including cancer, metabolic syndrome, cardiovascular disease, and neurodegenerative diseases, is discussed. Also presented are some related issues, such as the bioavailability of tea active components, the new formulations of tea polyphenols, and the safety for consumers of dietary supplements containing tea polyphenols.

Volatiles emitted from tea plants infested by Ectropis obliqua larvae are attractive to conspecific moths.

Herbivore-induced plant volatiles have been reported to play a role in the host-searching behavior of herbivores. However, next to nothing is known about the effect of volatiles emitted from tea plants infested by Ectropis obliqua larvae on the behavior of conspecific adults. Here, we found that tea plants infested by E. obliqua caterpillars for 24 h were more attractive to both virgin male and female E. obliqua adults than were intact, uninfested tea plants; moreover, mated female E. obliqua moths were more attracted by infested tea plants and preferentially oviposited on these plants, whereas male moths were repelled by infested plants once they had mated. Volatile analysis revealed that the herbivore infestation dramatically increased the emission of volatiles. Among these volatiles, 17 compounds elicited antennal responses from both male and female virginal moths. Using a Y-tube olfactometer, we found that 3 of the 17 chemicals, benzyl alcohol, (Z)-3-hexenyl hexanoate, and (Z)-3-hexenal, were attractive, but two compounds, linalool and benzyl nitril, were repellent to virgin male and female moths. One chemical, (Z)-3-hexenyl acetate, was attractive only to virgin males. Mated females were attracted by three compounds, (Z)-3-hexenyl hexanoate, (Z)-3-hexenyl acetate, and (Z)-3-hexenal; whereas mated males were repelled by (Z)-3-hexenol. The findings provide new insights into the interaction between tea plants and the herbivores, and may help scientists develop new measures with which to control E. obliqua.

Aroma changes of black tea prepared from methyl jasmonate treated tea plants.

Methyl jasmonate (MeJA) was widely applied in promoting food quality. Aroma is one of the key indicators in judging the quality of tea. This study examined the effect of exogenous MeJA treatment on tea aroma. The aroma components in black tea prepared from MeJA-treated fresh tea leaves were extracted using headspace solid-phase microextraction (HS-SPME) and were analyzed using gas chromatography-mass spectrometry (GC-MS) and GC-olfactometry (GC-O). Forty-five volatile compounds were identified. The results revealed that the MeJA-treated black tea had higher levels of terpene alcohols and hexenyl esters than the untreated tea. Moreover, several newly components, including copaene, cubenol, and indole, were induced by the MeJA treatment. The activities of polyphenol oxidase and ß-glucosidase in fresh tea leaves changed after the MeJA treatment. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the gene expression levels of polyphenol oxidase and ß-primeverosidase were upregulated by two and three folds, respectively, by the MeJA treatment (P<0.01); however, the gene expression of ß-glucosidase was downregulated to a half level. In general, the aroma quality of the MeJA-treated black tea was clearly improved.

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.

High-throughput analytical techniques for multiresidue, multiclass determination of 653 pesticides and chemical pollutants in tea--Part III: Evaluation of the cleanup efficiency of an SPE cartridge newly developed for multiresidues in tea.

A comparative study was conducted over three stages on the cleanup efficiency of SPE cartridge Cleanert TPT, newly developed for multigroups of pesticide residues in tea. In Stage I, different SPE cartridges C18, graphite carbon black (GCB), primary secondary amine (PSA), and amino (NH2) were purchased and combined into 12 different sequences. Through the comparative test on cleanup efficiency of 84 representative pesticides in tea, Envi-Carb GCB + PSA with a good cleanup effect was selected. In Stage II, GC/MS test results from the comparative study of the extraction efficiency of 201 pesticides spiked into green tea and Woolong tea with Cleanert TPT and Envi-Carb + PSA SPE showed that average recoveries fell within 70-110% and RSD <20% for 193 and 184 pesticides, respectively, for green tea, accounting for 96.0 and 91.0% of the total number, respectively. GC/MS/MS test results also found 193 and 184 pesticides, respectively, meeting the recovery and RSD conditions, accounting for 96.0 and 91.5%, respectively, of the total number. For Woolong tea samples, GC/MS results showed that with Cleanert TPT and Envi-Carb + PSA SPE for cleanup, there were 192 and 177 pesticides, respectively, meeting the conditions, accounting for 95.5 and 88.1% of the total number, respectively. GC/MS/MS results demonstrated that there were 195 and 184 pesticides, respectively, meeting the conditions, accounting for 97.0 and 91.5% of the total number, respectively. It was seen that Cleanert TPT was superior to Envi-Carb + PSA in cleanup efficiency, whether for green or Woolong tea samples, or GC/MS or GC/MS/MS determination. In Stage III, 61104 results of the average content value of pesticides and RSD (two teas xtwo Youden pair concentrations x two kinds of SPE cartridges x two instruments x 19 tests x 201 pesticides) were derived from the 19 times stability tests over 3 months by paralleling three samples every 5 days via two instruments with two kinds of SPE cartridges for cleanup, respectively, against Youden Pair samples of the 201 incurred pesticides from green and Woolong teas. The statistical analysis found that detected values from the target pesticides of the incurred Youden pair samples showed no marked differences with cleanup by either Cleanert TPT or Envi-Carb + PSA, whether for green or Woolong tea, or G/IMS or G/IM/IMS. The test results using the two aforementioned kinds of SPE cleanup for above 93% pesticides had a tolerance less than 15%, which testifies that both cartridge cleanups met the requirement for pesticide residue analysis.

Regurgitant derived from the tea geometrid Ectropis obliqua suppresses wound-induced polyphenol oxidases activity in tea plants.

Polyphenol oxidases (PPOs) have been reported to play an important role in protecting plants from attack by herbivores. However, little is known about their role in tea. Here, we investigated the effect of PPOs on interactions between tea plants and the tea geometrid Ectropis obliqua, one of the most important insect pests of tea. Jasmonic acid (JA) treatment resulted in increases in PPO activity, and the effect of JA was dose dependent. Ectropis obliqua caterpillars grew and developed more slowly on JA-treated tea plants than on control plants, and larval weight gains depended on the JA dosage. Artificial diet complemented with PPOs reduced the growth and survival rate of E. obliqua caterpillars, and there was a negative relationship between PPO level and larval growth and survival. Unlike mechanical wounding, which is an effective inducer of tea plant PPO activity, wounding plus the herbivore regurgitant or herbivore infestation suppressed the wound-induced PPO activities, especially at 4 days after treatment. These results suggest that PPOs are an important anti-herbivore factor in tea plants, defending them against E. obliqua larvae, and that E. obliqua larvae have evolved to elude the tea plant's defense by inhibiting the production of PPOs.

The tea weevil, Myllocerinus aurolineatus, is attracted to volatiles induced by conspecifics.

The tea weevil, Myllocerinus aurolineatus (Voss) (Coleoptera: Curculionidae), is a leaf-feeding pest of Camellia sinensis (O.Ktze.) with aggregative behaviors that can seriously reduce tea yield and quality. Although herbivore-induced host plant volatiles have been shown to attract conspecific individuals of some beetle pests, especially members of the Chrysomelidae family, little is known about the volatiles emitted from tea plants infested by M. aurolineatus adults and their roles in mediating interactions between conspecifics. The results of behavioral bioassays revealed that volatile compounds emitted from tea plants infested by M. aurolineatus were attractive to conspecific weevils. Volatile analyses showed that infestations dramatically increased the emission of volatiles, (Z)-3-hexenal, (Z)-3-hexenol, (E)-beta-ocimene, linalool, phenylethyl alcohol, benzyl nitrile, indole, (E, E)-alpha-farnesene, (E)-nerolidol, and 31 other compounds. Among the induced volatiles, 12 chemicals, including gamma-terpinene, benzyl alcohol, (Z)-3-hexenyl acetate, myrcene, benzaldehyde, (Z)-3-hexenal, and (E, E)-alpha-farnesene, elicited antennal responses from both sexes of the herbivore, whereas (E)-beta-ocimene elicited antennal responses only from males. Using a Y-tube olfactometer, we found that six of the 13 chemicals, gamma-terpinene, benzyl alcohol, (Z)-3-hexenyl acetate, myrcene, benzaldehyde, and (Z)-3-hexenal, were attractive to both males and females; two chemicals, (E/Z)-beta-ocimene and (E, E)-alpha-farnesene, were attractive only to males; and four chemicals, (E)-4,8-dimethyl-1,3,7-nonatriene, phenylethyl alcohol, linalool, and (Z)-3-hexenol, were attractive only to females. The findings provide new insights into the interactions between tea plants and their herbivores, and may help scientists develop new strategies for controlling the herbivore.