Secondary metabolites in host pears defense against two fruit borers and cytochrome-P450-mediated counter-defense.

Herbivorous insects have evolved metabolic strategies to survive the challenges posed by plant secondary metabolites (SMs). This study reports an exploration of SMs present in pears, which serve as a defense against invasive Cydia pomonella and native Grapholita molesta and their counter-defense response. The feeding preferences of fruit borers are influenced by the softening of two pear varieties as they ripen. The content of SMs, such as quercetin and rutin, increases due to feeding by fruit borers. Notably, quercetin levels only increase after C. pomonella feeding. The consumption of SMs affects the growth of fruit borer population differently, potentially due to the activation of P450 genes by SMs. These two fruit borers are equipped with specific P450 enzymes that specialize in metabolizing quercetin and rutin, enabling them to adapt to these SMs in their host fruits. These findings provide valuable insights into the coevolution of plants and herbivorous insects.

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.

Identification of Candidate Genes Associated with Type-II Sex Pheromone Biosynthesis in the Tea Geometrid (Ectropis obliqua) (Lepidoptera: Geometridae).

Ectropis obliqua, a notorious tea pest, produces a Type-II sex pheromone blend for mate communication. This blend contains (Z,Z,Z)-3,6,9-octadecatriene, (Z,Z)-3,9-cis-6,7-epoxy-octadecadiene, and (Z,Z)-3,9-cis-6,7-epoxy-nonadecadiene. To elucidate the genes related to the biosynthesis of these sex pheromone components, transcriptome sequencing of the female E. obliqua pheromone gland and the abdomen without pheromone gland was performed. Comparative RNAseq analyses identified 52 putative genes, including 7 fatty acyl-CoA elongases (ELOs), 9 fatty acyl-CoA reductases (FARs), 1 decarbonylase (DEC), 3 lipophorins (LIPs), and 32 cytochrome P450 enzymes (CYPs). Tissue expression profiles revealed that two ELOs (ELO3 and ELO5), two FARs (FAR2 and FAR9), one DEC (CYP4G173), and one LIP (LIP1) displayed either abdomen-centric or -specific expression, suggesting potential roles in sex pheromone biosynthesis within the oenocytes of E. obliqua. Furthermore, the tissue expression patterns, combined with phylogenetic analysis, showed that CYP340BD1, which was expressed specifically and predominantly only in the pheromone gland, was clustered with the previously reported epoxidases, highlighting its potential role in the epoxidation of the unsaturated polytriene sex pheromone components. Collectively, our research provides valuable insights into the genes linked to sex pheromone biosynthesis.

Identification, synthesis, and field evaluation of components of the female-produced sex pheromone of Helopeltis cinchonae (Hemiptera: Miridae), an emerging pest of tea.

BACKGROUND: Helopeltis cinchonae (Hemiptera: Miridae) is a major pest of tea plantations in Asia. Conventional control of pests with pesticides is unsustainable. Therefore, safe and eco-friendly alternatives, such as pheromones, are required to manage the pest. RESULTS: In gas chromatography-electroantennographic detection (GC-EAD) analysis of whole-body extracts of virgin female H. cinchonae, two compounds elicited electroantennogram (EAG) responses from male antennae. These were identified as hexyl (R)-3-acetoxybutyrate and (R)-1-acetoxy-5-butyroxyhexane using gas chromatography-mass spectrometry (GC-MS) analysis compared to synthetic compounds. This is the first study to report 1-acetoxy-5-butyroxyhexane as an insect pheromone component. The synthetic compounds elicited dose-dependent EAG responses from the antennae of male H. cinchonae. In two field trapping experiments, the individual compounds were highly attractive to male H. cinchonae when dispensed from polyethylene vials. However, higher catches were obtained with blends of the two compounds in a 1:10 ratio. The blend of racemic compounds was as attractive as the blend of (R)-enantiomers. CONCLUSIONS: We reported that 1-acetoxy-5-butyroxyhexane and hexyl 3-acetoxybutyrate are components of the female-produced sex pheromone of H. cinchonae, but further work is required on the blend and loading of pheromone and on trap design to provide an optimized system for monitoring and control of this pest. The results may also facilitate the identification of the pheromones of other Helopeltis species, which are major pests in many crops. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Transcriptome-Wide Identification of Cytochrome P450s in Tea Black Tussock Moth (Dasychira baibarana) and Candidate Genes Involved in Type-II Sex Pheromone Biosynthesis.

The tea black tussock moth (Dasychira baibarana), a devastating pest in Chinese tea plantations, uses a ternary Type-II pheromone blend containing (3Z,6Z)-cis-9,10-epoxyhenicosa-3,6-diene (Z3,Z6,epo9-21:H), (3Z,6Z,11E)-cis-9,10-epoxyhenicosa-3,6,11-triene (Z3,Z6,epo9,E11-21:H), and (3Z,6Z)-henicosa-3,6-dien-11-one (Z3,Z6-21:11-one) for mate communication. To elucidate the P450 candidates associated with the biosynthesis of these sex pheromone components, we sequenced the female D. baibarana pheromone gland and the abdomen excluding the pheromone gland. A total of 75 DbP450s were identified. Function annotation suggested six CYPs were orthologous genes that are linked to molting hormone metabolism, and eight antennae specifically and significantly up-regulated CYPs may play roles in odorant processing. Based on a combination of comparative RNAseq, phylogenetic, and tissue expression pattern analysis, one CYP4G with abdomen specifically predominant expression pattern was likely to be the P450 decarbonylase, while the pheromone-gland specifically and most abundant CYP341B65 was the most promising epoxidase candidate for the D. baibarana sex pheromone biosynthesis. Collectively, our research laid a valuable basis not only for further functional elucidation of the candidate P450 decarbonylase and epoxidase for the sex pheromone biosynthesis but also for understanding the physiological functions and functional diversity of the CYP gene superfamily in the D. baibarana.

Identification of aggregation pheromones released by the stick tea thrips (Dendrothrips minowai) larvae and their application for controlling thrips in tea plantations.

BACKGROUND: The stick tea thrips, Dendrothrips minowai Priesner, is one of the most important sucking pests that seriously infest tea plants (Camellia sinensis) in China. Given that D. minowai exhibit aggregation behaviors in tea plantations, this study evaluated the potential of aggregation pheromones for their control. RESULTS: H-tube olfactometer assays showed that D. minowai larvae, adult females and adult males were significantly attracted to larvae rather than adult females and males under laboratory conditions. Subsequent gas chromatography-mass spectrometry analysis of volatiles from larvae, identified two larva-specific components: dodecyl acetate and tetradecyl acetate. Electrophysiological and behavioral experiments confirmed the positive response of females and males to dodecyl acetate, tetradecyl acetate, and their blend (1:1.5). Deployment of these aggregation pheromones on sticky traps resulted in a 1.2- to 3.0-fold increase in the capture of D. minowai adults compared with control traps. In addition, deployment of sticky traps baited with these aggregation pheromones within tea plantations resulted in a noteworthy reduction in the population of adult thrips per 100 leaves, 10 days following trap deployment. The reduction ranged from 29% to 59%, in comparison with the control. CONCLUSION: D. minowai larvae produce aggregation pheromones, dodecyl acetate and tetradecyl acetate, that can be useful for controlling tea thrips. © 2023 Society of Chemical Industry.

Activity Patterns, Population Dynamics, and Spatial Distribution of the Stick Tea Thrips, Dendrothrips minowai, in Tea Plantations.

The stick tea thrips, D. minowai Priesner (Thysanoptera: Thripidae), is one of the most economically significant thrips pests of tea (Camellia sinensis (L.) O. Ktze.) in China. Here, we sampled D. minowai in tea plantations from 2019 to 2022 to characterize its activity patterns, population dynamics, and spatial distribution. A large proportion of D. minowai individuals were caught in traps placed at heights ranging from 5 cm below to 25 cm above the position of tender leaves at the top of the tea plant, and the greatest number of individuals were captured at a height of 10 cm from the position of tender leaves at the top of the tea plant. Thrips were most abundant from 10:00 to 16:00 h in the spring and from 06:00 to 10:00 h and from 16:00 to 20:00 h on sunny days in the summer. The spatial distribution of D. minowai females and nymphs was aggregated on leaves according to Taylor's power law (females: R2 = 0.92, b = 1.69 > 1; nymphs: R2 = 0.91, b = 2.29 > 1) and Lloyd's patchiness index (females and nymphs: C > 1, Ca > 0, I > 0, M*/m > 1). The D. minowai population was dominated by females, and male density increased in June. Adult thrips overwintered on the bottom leaves, and they were most abundant from April to June and from August to October. Our findings will aid efforts to control D. minowai populations.

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 and Field Evaluation of Sex Pheromone Components and Its Antagonist Produced by a Major Tea Pest, Archips strojny (Lepidoptera: Tortricidae).

Pesticide application is the only known control method for the tea tortrix Archips strojny (Lepidoptera: Tortricidae), which is a major pest of spring tea in China. To develop sex pheromone-based, environmentally safe control strategies, here we identified the sex pheromone components of this species. The male moths' antennae responded electrophysiologically to two compounds in female pheromone gland extracts. Gas chromatography-mass spectrometry analysis indicated that the two bioactive compounds were (Z)-11-tetradecenyl acetate (Z11-14:Ac) and (Z)-11-tetradecenyl alcohol (Z11-14:OH). Field trapping assays showed that lures baited with only the major component Z11-14:Ac were the most attractive to male moths, and the attractiveness decreased significantly when the lure was impregnated with increased relative ratios of the minor component Z11-14:OH. Our study demonstrated that Z11-14:Ac was the major attractant in the A. strojny sex pheromone, and the minor component Z11-14:OH seemed to serve as an antagonist. The results indicate that lures baited with 1 mg of Z11-14:Ac could be used as a monitoring or mass trapping tool for A. strojny management in Chinese tea plantations. Furthermore, Z11-14:Ac was identified as a common sex pheromone attractant of nine Archips species; these results lay the foundation for developing mating disruption techniques that target multiple leafroller pests.

Enhanced volatile emissions and anti-herbivore functions mediated by the synergism between jasmonic acid and salicylic acid pathways in tea plants.

The interaction between jasmonic acid (JA) and salicylic acid (SA) pathways, which affects plant stress resistance, is mainly considered to be antagonistic. Using an established theoretical model, we investigated how tea plant (Camellia sinensis) volatiles induced by exogenous elicitors of the JA and SA pathways are affected by the sequence of elicitor application, elicitor identity, and the applied concentrations. We also examined the effects of the volatiles mediated by the JA-SA synergistic interaction on the behaviors of a tea leaf-chewing herbivore (Ectropis grisescens) and its parasitic wasp (Apanteles sp.). The JA and SA pathway interactions were almost always reciprocally synergistic when the two pathways were elicited at different times, except at high JA elicitor concentrations. However, the JA pathway antagonized the SA pathway when they were elicited simultaneously. The elicitor identity affected the degree of JA-SA interaction. The volatiles induced by the JA pathway in the JA-SA reciprocal synergism treatments included up to 11 additional compounds and the total amount of volatiles was up to 7.9-fold higher. Similarly, the amount of emitted volatiles induced by the SA pathway in the reciprocal synergism treatments increased by up to 4.2-fold. Compared with the volatiles induced by either pathway, the enriched volatiles induced by the JA-SA reciprocal synergism similarly repelled E. grisescens, but attracted Apanteles sp. more strongly. Thus, non-simultaneous activation is important for optimizing the JA-SA reciprocal synergism. This reciprocal synergism enables plants to induce multifarious responses, leading to increased biotic stress resistance.

Evaluation of Selected Plant Volatiles as Attractants for the Stick Tea Thrip Dendrothrips minowai in the Laboratory and Tea Plantation.

The stick tea thrip (Dendrothrips minowai Priesner) is the main pest thrip in tea (Camellia sinensis) plantations in China, and seriously affects the quality and yield of tea. Plant-derived semiochemicals provide an alternative to pheromones as lures and these compounds possess powerful attractiveness. In this study, we selected 20 non-pheromone semiochemicals, including compounds that have been reported to attract other thrips and some volatiles emitted from tea plants as the potential attractant components for D. minowai. In electroantennogram (EAG) assays, 10 synthetic compounds (p-anisaldehyde, 3-methyl butanal, (E)-ß-ocimene, farnesene, nonanal, eugenol, (+)-α-pinene, limonene, (-)-α-pinene, and γ-terpinene) elicited significant antennal responses in female D. minowai. In addition, a two-choice H-tube olfactometer bioassay showed that D. minowai displayed significant positive responses to eight compound dilutions (p-anisaldehyde, eugenol, farnesene, methyl benzoate, 3-methyl butanal, (E)-ß-ocimene, (-)-α-pinene, and (+)-α-pinene) when compared with the solvent control at both 1 and 2 h. Moreover, γ-terpinene exhibited a significantly deterrent effect on D. minowai. Finally, trap catches of four compounds (p-anisaldehyde, eugenol, farnesene, and 3-methyl butanal, respectively) significantly increase in tea plantations. Among these, the maximum number of D. minowai collected by blue sticky traps baited with p-anisaldehyde was 7.7 times higher than the control. In conclusion, p-anisaldehyde, eugenol, farnesene, and 3-methyl butanal could significantly attract D. minowai in the laboratory and under field conditions, suggesting considerable potential as commercial attractants to control D. minowai populations.

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.

Variation in the ratio of compounds in a plant volatile blend during transmission by wind.

For plant volatiles to mediate interactions in tritrophic systems, they must convey accurate and reliable information to insects. However, it is unknown whether the ratio of compounds in plant volatile blends remains stable during wind transmission. In this study, volatiles released from an odor source were collected at different points in a wind tunnel and analyzed. The variation in the amounts of volatiles collected at different points formed a rough cone shape. The amounts of volatiles collected tended to decrease with increasing distance from the odor source. Principal component analyses showed that the volatile profiles were dissimilar among different collection points. The profiles of volatiles collected nearest the odor source were the most similar to the released odor. Higher wind speed resulted in a clearer spatial distribution of volatile compounds. Thus, variations in the ratios of compounds in odor plumes exist even during transport over short distances.

Involvement of CYP347W1 in neurotoxin 3-nitropropionic acid-based chemical defense in mustard leaf beetle Phaedon cochleariae.

Chrysomelina beetlesstore 3-nitropropionic acid in form of a pretoxin, isoxazolin-5-one glucoside-conjugated ester, to protect themselves against predators. Here we identified a cytochrome P450 monooxygenase, CYP347W1, to be involved in the production of the 3-nitropropionic acid moiety of the isoxazolin-5-one glucoside ester. Knocking down CYP347W1 led to a significant depletion in the concentration of the isoxazolin-5-one glucoside ester and an increase in the concentration of the isoxazolin-5-one glucoside in the larval hemolymph. Enzyme assays with the heterologously expressed CYP347W1 showed free ß-alanine was not the direct substrate. Homology modeling indicated that ß-alanine-CoA ester can fit into CYP347W1's active site. Furthermore, we proved that Phaedon cochleariae eggs are not able to de novo synthesize 3-NPA, although both isoxazolin-5-one glucoside and its 3-NPA-conjugated ester are present in the eggs. These results provide direct evidence for the involvement of CYP347W1 in the biosynthesis of a P. cochleariae chemical defense compound.

A cytochrome P450 from the mustard leaf beetles hydroxylates geraniol, a key step in iridoid biosynthesis.

Larvae of the leaf beetle Phaedon cochleariae synthesize the iridoid chysomelidial via the mevalonate pathway to repel predators. The normal terpenoid biosynthesis is integrated into the dedicated defensive pathway by the ω-hydroxylation of geraniol to (2E,6E)-2,6-dimethylocta-2,6-diene-1,8-diol (ω-OH-geraniol). Here we identify and characterize the P450 monooxygenase CYP6BH5 as the geraniol hydroxylase using integrated transcriptomics, proteomics and RNA interference (RNAi). In the fat body, 73 cytochrome P450s were identified, and CYP6BH5 was among those that were expressed specifically in fat body. Double stranded RNA mediated knockdown of CYP6BH5 led to a significant reduction of ω-hydroxygeraniol glucoside in the hemolymph and, later, of the chrysomelidial in the defensive secretion. Heterologously expressed CYP6BH5 converted geraniol to ω-OH-geraniol. In addition to geraniol, CYP6BH5 also catalyzes hydroxylation of other monoterpenols, such as nerol and citronellol to the corresponding α,ω-dihydroxy compounds.