Isolation and expression of cytochrome P450 genes in the antennae and gut of pine beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) following exposure to host monoterpenes

Bark beetles oxidize the defensive monoterpenes of their host trees both to detoxify them and convert them into components of their pheromone system. This oxidation is catalyzed by cytochrome P450 enzymes and occurs in different tissues of the insect, including the gut (i.e., the site where the beetle's pheromones are produced and accumulated) and the antennae (i.e., the olfactory organs used for perception of airborne defensive monoterpenes as well as other host-associated compounds and pheromones). We identified ten new CYP genes in the pine beetle Dendroctonus rhizophagus in either antennae or gut tissue after stimulation with the vapors of major host monoterpenes α-pinene, β-pinene and 3-carene. Five genes belong to the CYP4 family, four to the CYP6 family and one to the CYP9 family. Differential expression of almost all of the CYP genes was observed between sexes, and within these significant differences among time, stimuli, anatomical region, and their interactions were found upon exposure to host monoterpenes. Increased expression of cytochrome P450 genes suggests that they play a role in the detoxification of monoterpenes released by this insect's host trees.     

Diversity and expression of P450 genes from Dendroctonus valens LeConte (Curculionidae: Scolytinae) in response to different kairomones

Bark beetles (Curculionidae: Scolytinae) are major cause of woody plants death in the world. They colonize the stem and other parts of trees recognizing host-produced specific compounds (kairomones) and insect pheromones. Bark beetle's antennae and alimentary canal participate in the host selection identifying chemical compounds produced by trees and insects, and also in the metabolism and detoxification of these compounds. The red turpentine beetle (RTB), Dendroctonus valens LeConte, is an unaggressive species that colonize > 40 pine species (Pinaceae) in North and Central America. Several studies suggest that bark beetle cytochrome P450 enzymes are involved in monoterpene oxidation. In this study we identified by means of PCR, cloning, sequencing, and bioinformatic analysis, eleven full-length genes: five CYP4, four CYP6, and two CYP9 in the antennae and gut region of RTB, after stimulation with vapors of monoterpenes: (±)-α-pinene, (R)-(+)-α-pinene, (S)-(?)-β-pinene, (S)-(?)-α-pinene and (+)-3-carene; pine trees volatiles used by RTB as kairomones. The recovered cDNA of these genes vary from 1.5 kb to 1.8 kb and the open frame encodes from 496 to 562 amino acid proteins. The bioinformatic analysis suggests that the majority of P450 proteins encoded by these genes are membrane anchored in the endoplasmic reticulum. RT-qPCR assays showed differential expression of all CYP genes between male and female. The gene expression was dependent of monoterpenes and exposure time, with some of them sex, antennae and gut region specific. Significant differences among monoterpenes, gut region, antennae and exposure time were found. Our results suggest that some of these genes may be involved in the detoxification process of these compounds during tree colonization.     

Functional characterization of myrcene hydroxylases from two geographically distinct Ips pini populations

Ips pini bark beetles use myrcene hydroxylases to produce the aggregation pheromone component, ipsdienol, from myrcene. The enantiomeric ratio of pheromonal ipsdienol is an important prezygotic mating isolation mechanism of I. pini and differs among geographically distinct populations. We explored the substrate and product ranges of myrcene hydroxylases (CYP9T2 and CYP9T3) from reproductively-isolated western and eastern I. pini. The two cytochromes P450 share 94% amino acid identity. CYP9T2 mRNA levels were not induced in adults exposed to myrcene-saturated atmosphere. Functional assays of recombinant enzymes showed both hydroxylated myrcene, (+)- and (?)-α-pinene, 3-carene, and R-(+)-limonene, but not α-phellandrene, (?)-β-pinene, γ-terpinene, or terpinolene, with evidence that CYP9T2 strongly preferred myrcene over other substrates. They differed in the enantiomeric ratios of ipsdienol produced from myrcene, and in the products resulting from different α-pinene enantiomers. These data provide new information regarding bark beetle pheromone evolution and factors affecting cytochrome P450 structure–function relationships.     

Ophiostomatoid fungi can emit the bark beetle pheromone verbenone and other semiochemicals in media amended with various pine chemicals and beetle-released compounds

Fungal volatile compounds can mediate fungal-insect interactions. Whether fungi can emit insect pheromones and how volatile chemicals change in response to chemicals the fungi naturally encounter is poorly understood. We analyzed volatiles emitted by Grosmannia clavigera (symbiont of the mountain pine beetle) and Ophiostoma ips (symbiont of the pine engraver beetle) growing in liquid media amended with compounds that the fungi naturally encounter: (−)-α-pinene, (+)-α-pinene, (−)-trans-verbenol, verbenone, or ipsdienol. Nine volatile compounds were identified among the fungal and amendment treatments. Volatiles qualitatively and quantitatively differed between fungal species and among amendment treatments. The bark beetle anti-aggregation pheromone (−)-verbenone was detected from both fungi growing in (−)-trans-verbenol-amended media. G. clavigera and O. ips can emit beetle pheromones and other beetle semiochemicals, suggesting that ophiostomatoid fungi could contribute to the chemical ecology of bark beetles. However, such contributions could be modulated by the presence of other environmental chemicals.     

Red turpentine beetle primary attraction to β-pinene or 3-carene (with and without ethanol) varies in western US pine forests

1. Lure attraction strength for red turpentine beetle, Dendroctonus valens (Coleoptera: Curculionidae: Scolytinae) observed previously in US Pacific Northwest ponderosa pine forests is (−)-β-pinene+ethanol > (+)-3-carene+ethanol, but untested elsewhere in its western US range. Thus, both were tested with (−)-β-pinene, (+)-3-carene, ethanol, and a blank in Oregon and California sites burned by wildfire, whereas in Arizona the first four lures were tested in a thinned-unburned site.
2. The D. valens responses in burned Oregon and California sites were similar, (−)-β-pinene+ethanol > (−)-β-pinene > 3-carene = 3-carene+ethanol > ethanol > blank, whereas in the cut-unburned Arizona site it was 3-carene+ethanol > 3-carene = (−)-β-pinene+ethanol > (−)-β-pinene. Whether this variation was influenced by beetle genetic differences, or chemical and physical parameters in the different environments and remaining stressed host resources 1-year post disturbance warrants additional study.
3. Responses to (−)-β-pinene varied, from a stronger attractant than (+)-3-carene in Oregon and California, to a weaker lure than (+)-3-carene in Arizona. This (−)-β-pinene variability was minimized when released in combination with ethanol, making (−)-β-pinene+ethanol the most consistent attractant of those tested across the three states, and a reliable lure for detection, monitoring, and management projects for D. valens in western US pine forests.

     

β-Pinene + ethanol attracts more red turpentine beetles than carene+ ethanol,with or without traces of frontalin,at prescribed burn or thinned sites

1. Red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae: Scolytinae), previously responded more strongly to (−)-β-pinene + ethanol than (+)-3-carene + ethanol lures at sites burned the prior year by wildfire in Oregon and northeastern California, whereas at a thinned-unburned Arizona site (+)-3-carene + ethanol was the stronger attractant. This discrepancy was further examined to tease apart whether D. valens attraction varies by region or previous forest disturbance types.
2. Here, (−)-β-pinene + ethanol and (+)-3-carene + ethanol lures were tested in pine stands at two Oregon sites disturbed the previous year by a prescribed burn or thinning only. Both lures were tested also with or without trace amounts of the pheromone frontalin, as its presence enhanced attractions in China but had not been tested in North America.
3. At both sites, regardless of prior forest disturbance, (−)-β-pinene + ethanol lures attracted the most beetles. Lures releasing trace frontalin attracted more beetles than their corresponding lures without it at both sites, except in one case.
4. Overall, previous year disturbances from disparate management treatments had minimal influence on lure attraction to D. valens. For detection, monitoring or management (−)-β-pinene + ethanol + frontalin in trace amounts attracts the most beetles of lures tested to date in Pacific Northwest pine forests.

     

Influence of sex,maturity and host substances on pheromones in the guts of the bark beetles, Ips paraconfusus and Dendroctonus brevicomis

Pheromones and metabolites of host (ponderosa pine) compounds were found in association with the hindgut of both naturally fed and of non-fed, host vapour-exposed bark beetles, Ips paraconfusus and Dendroctonus brevicomis. Much smaller amounts were found in the corresponding heads and mid guts. Sex-specific differences in content of pheromones were observed as in earlier studies. Exposure of I. paraconfusus to vapours of a pheromone component, ipsenol and other monoterpene alcohols resulted in their accumulation in the hindgut but relatively very low amounts in the head. The possible sites of pheromone biosynthesis are discussed. Exposure of male I. paraconfusus to vapours of host compounds, myrcene and α-pinene, revealed that immature adults do not produce the pheromone components, ipsenol and ipsdienol, as mature adults do while both immature and mature sexes produced another pheromone component, cis-verbenol, as well as trans-verbenol and myrtenol. Immature D. brevicomis adults did not contain pheromones until their exposure to vapours of (?)-α-pinene which caused production of trans-verbenol but only about 10% that of mature adults treated similarly. Verbenone, a male-produced inhibitory pheromone of D. brevicomis, apparently was not synthesized from (?)-α-pinene in females nor was its synthesis in males enhanced by exposure to this host compound.     

Development of lady beetle attractants from floral volatiles and other semiochemicals for the biological control of aphids

Aphids are among the most destructive phytophagous pests of host plants, because of their rapid reproduction, parthenogenesis, extensive crop damage, and the transmission of many plant viruses. Since lady beetles are important predatory natural enemies of aphids, developing lady beetle attractants to increase their field abundance is vital for aphid control. Floral volatiles and other semiochemicals are reportedly attractive to lady beetles. In this research, a total of 58 floral volatiles were tested by Y-tube olfactometer assays, among which 29 were highly attractive to both Harmonia axyridis (Coleoptera: Coccinellidae) and Coccinella septempunctata (Coleoptera: Coccinellidae). Meanwhile, the results of wind tunnel trials showed that only isoamyl acetate, α-humulene, trans-3-hexen-1-ol, methyl salicylate, and β-pinene lure these two species. Thereafter, 15 semiochemicals from pests, natural enemies, and pest-infested crops were mixed with the selected floral volatiles, to determine optimum formulations for attracting lady beetles through wind tunnels and further field trials. Eventually, formulas (1) α-humulene: β-pinene: methyl salicylate: trans-3-hexen-1-ol = 1:3:3:10; (5) α-humulene: β-pinene: methyl salicylate:1-nonanal = 1:3:3:10; (8) α-humulene: β-pinene: methyl salicylate: indole = 1:3:3:10 (1, 5, and 8 are labels used for the various formulations in field trials) were successfully verified to be attractive to lady beetles in both pumpkin and wheat fields. These formulations will be of great significance in developing integrated pest management strategies for aphid control.     

Two Herbivore-Induced Cytochrome P450 Enzymes CYP79D6 and CYP79D7 Catalyze the Formation of Volatile Aldoximes Involved in Poplar Defense

Aldoximes are known as floral and vegetative plant volatiles but also as biosynthetic intermediates for other plant defense compounds. While the cytochrome P450 monooxygenases (CYP) from the CYP79 family forming aldoximes as biosynthetic intermediates have been intensively studied, little is known about the enzymology of volatile aldoxime formation. We characterized two P450 enzymes, CYP79D6v3 and CYP79D7v2, which are involved in herbivore-induced aldoxime formation in western balsam poplar (Populus trichocarpa). Heterologous expression in Saccharomyces cerevisiae revealed that both enzymes produce a mixture of different aldoximes. Knockdown lines of CYP79D6/7 in gray poplar (Populus × canescens) exhibited a decreased emission of aldoximes, nitriles, and alcohols, emphasizing that the CYP79s catalyze the first step in the formation of a complex volatile blend. Aldoxime emission was found to be restricted to herbivore-damaged leaves and is closely correlated with CYP79D6 and CYP79D7 gene expression. The semi-volatile phenylacetaldoxime decreased survival and weight gain of gypsy moth (Lymantria dispar) caterpillars, suggesting that aldoximes may be involved in direct defense. The wide distribution of volatile aldoximes throughout the plant kingdom and the presence of CYP79 genes in all sequenced genomes of angiosperms suggest that volatile formation mediated by CYP79s is a general phenomenon in the plant kingdom.     

Identification,characterization, and expression of two novel P450 genes CYP6BQ21 and CYP6BQ22 from Dastarcus helophoroides (Coleoptera: Bothrideridae)

Chemical pesticides often applied to effectively control the long-horned beetles to protect the forests are reported to affect the non-target organisms adversely. Dastarcus helophoroides is an active natural enemy of long-horned beetles. Studying the molecular mechanism of P450 genes will help to elucidate the metabolic mechanism of pesticides in D. helophoroides to better coordinate the use of chemical and biological controls. In this study, two novel genes, CYP6BQ21, and CYP6BQ22 were successfully cloned from D. helophoroides using the rapid amplification of cDNA ends technique. The sequence and homology analyses indicated that CYP6BQ21 was highly similar to CYP6BQ1 from Tribolium castaneum, while CYP6BQ22 was closely related to CYP6BQ13 from T. castaneum. Gene expression patterns showed that CYP6BQ21 and CYP6BQ22 were specifically expressed in the adult stage of D. helophoroides. In addition, CYP6BQ21 and CYP6BQ22 were significantly expressed under the treatment of a high concentration of cypermethrin. Based on these findings, we proposed that CYP6BQ21 and CYP6BQ22 played possible roles in the development and pesticide stress of D. helophoroides. Our findings are an important first step in identifying and characterizing CYP6BQ21 and CYP6BQ22 from D. helophoroides, and lay the groundwork for future research into the role of these novel CYP6s in the regulation of pesticide resistance in D. helophoroides.     

Oxidation products of pinene in the bark beetle,Dendroctonus frontalis

Southern pine beetles were collected as they emerged from infested pine bolts and exposed in Petri dishes to α- and β-pinene for 20 hr. Changes in the volatile contents of the beetles' hindguts were detected by gas chromatography, and previously unidentified components were characterized by mass, i.r., and n.m.r. spectroscopy. A prominent compound in hindguts of both sexes before and after treatment was identified as myrtenol, and a less conspicuous, male-specific compound proved to be myrtenal. Upon exposure to α-pinene, males produced cis- and trans-verbenol, and 4-methyl-2-pentanol was found in both sexes. Compounds present after treatment with β-pinene vapour were identified as trans-pinocarveol in both sexes, and pinocarvone in the males.     

Geographic association and temporal variation of chemical and physical defense and leaf damage in Datura stramonium

The evolution of plant defense traits has traditionally been explained trough the “coevolutionary arms race” between plants and herbivores. According to this, specialist herbivores have evolved to cope effectively with the defensive traits of their host plants and may even use them as a cue for host location. We analyzed the geographic association between leaf trichomes, two tropane alkaloids (putative resistance traits), and leaf damage by herbivores in 28 populations of Datura stramonium in central Mexico. Since the specialist leaf beetles Epitrix parvula and Lema trilineata are the main herbivores of D. stramonium in central Mexico, we predicted a positive association between plant defense and leaf damage across populations. Also, if physical environmental conditions (temperature or precipitation) constrain the expression of plant defense, then the geographic variation in leaf damage should be explained partially by the interaction between defensive traits and environmental factors. Furthermore, we studied the temporal and spatial variation in leaf trichome density and leaf damage in five selected populations of D. stramonium sampled in two periods (1997 vs. 2007). We found a positive association between leaf trichomes density and atropine concentration with leaf damage across populations. The interaction between defensive traits and water availability in each locality had a significant effect on the geographic variation in leaf damage. Differences among populations in leaf trichome density are maintained over time. Our results indicate that local plant–herbivore interaction plays an important role in shaping the geographic and temporal variation in plant defense in D. stramonium.     

Functional characterization of the Tetranychus urticae CYP392A11, a cytochrome P450 that hydroxylates the METI acaricides cyenopyrafen and fenpyroximate

Cyenopyrafen is a Mitochondrial Electron Transport Inhibitor (METI) acaricide with a novel mode of action at complex II, which has been recently developed for the control of the spider mite Tetranychus urticae, a pest of eminent importance globally. However, some populations of T. urticae are cross-resistant to this molecule, and cyenopyrafen resistance can be readily selected in the lab. The cytochrome P450s genes CYP392A11 and CYP392A12 have been strongly associated with the phenotype. We expressed the CYP392A11 and the CYP392A12 genes with T. urticae cytochrome P450 reductase (CPR) in Escherichia coli. CYP392A12 was expressed predominately as an inactive form, witnessed by a peak at P420, despite optimization efforts on expression conditions. However, expression of CYP392A11 produced a functional enzyme, with high activity and preference for the substrates Luciferin-ME EGE and ethoxycoumarin. CYP392A11 catalyses the conversion of cyenopyrafen to a hydroxylated analogue (k_cat = 2.37 pmol/min/pmol P450), as well as the hydroxylation of fenpyroximate (k_cat = 1.85 pmol/min/pmol P450). In addition, transgenic expression of CYP392A11 in Drosophila melanogaster, in conjunction with TuCPR, confers significant levels of fenpyroximate resistance.The overexpression of CYP392A11 in multi-resistant T. urticae strains, not previously exposed to cyenopyrafen, which had been indicated by microarray studies, was confirmed by qPCR, and it was correlated with significant levels of cyenopyrafen and fenpyroximate cross-resistance. The implications of our findings for insecticide resistance management strategies are discussed.     

6种植物次生物质对斜纹夜蛾解毒酶活性的影响

草食性昆虫取食植物时遇到宿主植物中大量次生物质的化学防御,研究昆虫适应植物毒素的反防御策略具有重要的科学意义。分别添加0.01%肉桂酸、0.01%水杨酸、0.01%花椒毒素、0.02%槲皮素、0.05%黄酮和0.1%香豆素等6种植物次生物质的人工饲料饲养斜纹夜蛾(Spodoptera litura)五龄幼虫48 h后,测定斜纹夜蛾幼虫中肠和脂肪体中谷胱甘肽S-转移酶(GSTs)、羧酸酯酶(CarE)、P450的酶含量及头部乙酰胆碱酯酶(AChE)的活性,利用半定量RT-PCR检测中肠和脂肪体中CYP4M14和CYP4S9的基因表达水平。结果表明:取食肉桂酸和香豆素后,斜纹夜蛾中肠中CarE的酶活性分别提高了1.67和1.37倍,取食6种次生物质均能显著提高斜纹夜蛾脂肪体中GSTs酶活性。取食肉桂酸和香豆素48 h后,脂肪体中P450酶含量比对照增加2.93和14.50倍。取食肉桂酸、花椒毒素、槲皮素和香豆素后,斜纹夜蛾头部AchE酶活性与对照相比提高了1.53、1.80、2.36和1.56倍。6种次生物质均可诱导脂肪体中CYP4M14基因表达,槲皮素、肉桂酸和香豆素强烈诱导CYP4S9在脂肪体中表达。表明,斜纹夜蛾具有利用植物次生物质诱导其解毒酶的能力,进而提高其对毒素的抗性。