Biosynthesis of iridoid sex pheromones in aphids.

Iridoid monoterpenes, widely distributed in plants and insects, have many ecological functions. While the biosynthesis of iridoids has been extensively studied in plants, little is known about how insects synthesize these natural products. Here, we elucidated the biosynthesis of the iridoids cis-trans-nepetalactol and cis-trans-nepetalactone in the pea aphid Acyrthosiphon pisum (Harris), where they act as sex pheromones. The exclusive production of iridoids in hind legs of sexual female aphids allowed us to identify iridoid genes by searching for genes specifically expressed in this tissue. Biochemical characterization of candidate enzymes revealed that the iridoid pathway in aphids proceeds through the same sequence of intermediates as described for plants. The six identified aphid enzymes are unrelated to their counterparts in plants, conclusively demonstrating an independent evolution of the entire iridoid pathway in plants and insects. In contrast to the plant pathway, at least three of the aphid iridoid enzymes are likely membrane bound. We demonstrated that a lipid environment facilitates the cyclization of a reactive enol intermediate to the iridoid cyclopentanoid-pyran scaffold in vitro, suggesting that membranes are an essential component of the aphid iridoid pathway. Altogether, our discovery of this complex insect metabolic pathway establishes the genetic and biochemical basis for the formation of iridoid sex pheromones in aphids, and this discovery also serves as a foundation for understanding the convergent evolution of complex metabolic pathways between kingdoms.

Biology, Ecology, and Management of Flea Beetles in Brassica Crops.

Brassica vegetable and oilseed crops are attacked by several different flea beetle species (Chrysomelidae: Alticini). Over the past decades, most research has focused on two Phyllotreta species, Phyllotreta striolata and Phyllotreta cruciferae, which are major pests of oilseed rape in North America. More recently, and especially after the ban of neonicotinoids in the European Union, the cabbage stem flea beetle, Psylliodes chrysocephala, has become greatly important and is now considered to be the major pest of winter oilseed rape in Europe. The major challenges to flea beetle control are the prediction of population dynamics in the field, differential susceptibility to insecticides, and the lack of resistant plant cultivars and other economically viable alternative management strategies. At the same time, many fundamental aspects of flea beetle biology and ecology, which may be relevant for the development of sustainable control strategies, are not well understood. This review focuses on the interactions between flea beetles and plants and summarizes the literature on current management strategies with an emphasis on the potential for biological control in flea beetle management.

Escalation by duplication: Milkweed bug trumps Monarch butterfly.

The iconic Monarch butterfly is probably the best-known example of chemical defence against predation, as pictures of vomiting naive blue jays in countless textbooks vividly illustrate. Larvae of the butterfly take up toxic cardiac glycosides from their milkweed hostplants and carry them over to the adult stage. These compounds (cardiotonic steroids, including cardenolides and bufadienolides) inhibit the animal transmembrane sodium-potassium ATPase (Na,K-ATPase), but the Monarch enzyme resists this inhibition thanks to amino acid substitutions in its catalytic alpha-subunit. Some birds also have substitutions and can feast on cardiac glycoside-sequestering insects with impunity. A flurry of recent work has shown how the alpha-subunit gene has been duplicated multiple times in separate insect lineages specializing in cardiac glycoside-producing plants. In this issue of Molecular Ecology, Herbertz et al. toss the beta-subunit into the mix, by expressing all nine combinations of three alpha- and three beta-subunits of the milkweed bug Na,K-ATPase and testing their response to a cardenolide from the hostplant. The findings suggest that the diversification and subfunctionalization of genes allow milkweed bugs to balance trade-offs between resistance towards sequestered host plant toxins that protect the bugs from predators, and physiological costs in terms of Na,K-ATPase activity.

Sequestration of Plant Defense Compounds by Insects: From Mechanisms to Insect-Plant Coevolution.

Plant defense compounds play a key role in the evolution of insect-plant associations by selecting for behavioral, morphological, and physiological insect adaptations. Sequestration, the ability of herbivorous insects to accumulate plant defense compounds to gain a fitness advantage, represents a complex syndrome of adaptations that has evolved in all major lineages of herbivorous insects and involves various classes of plant defense compounds. In this article, we review progress in understanding how insects selectively accumulate plant defense metabolites and how the evolution of specific resistance mechanisms to these defense compounds enables sequestration. These mechanistic considerations are further integrated into the concept of insect-plant coevolution. Comparative genome and transcriptome analyses, combined with approaches based on analytical chemistry that are centered in phylogenetic frameworks, will help to reveal adaptations underlying the sequestration syndrome, which is essential to understanding the influence of sequestration on insect-plant coevolution.

Gut microbiota degrades toxic isothiocyanates in a flea beetle pest.

Microbial symbionts of herbivorous insects have been suggested to aid in the detoxification of plant defense compounds; however, quantitative studies on microbial contribution to plant toxin degradation remain scarce. Here, we demonstrate microbiome-mediated degradation of plant-derived toxic isothiocyanates in the cabbage stem flea beetle Psylliodes chrysocephala, a major pest of oilseed rape. Suppression of microbiota in antibiotic-fed beetles resulted in up to 11.3-fold higher levels of unmetabolized isothiocyanates compared to control beetles but did not affect other known detoxification pathways in P. chrysocephala. We characterized the microbiome of laboratory-reared and field-collected insects using 16S rRNA amplicon sequencing and isolated bacteria belonging to the three core genera Pantoea, Acinetobacter and Pseudomonas. Only Pantoea isolates rapidly degraded isothiocyanates in vitro, and restored isothiocyanate degradation in vivo when reintroduced in antibiotic-fed beetles. Pantoea was consistently present across beetle life stages and in field and lab populations. In addition, Pantoea was detected in undamaged tissues of the host plant Brassica rapa, indicating that P. chrysocephala could possibly acquire an isothiocyanate detoxifying bacterium through their diet. Our results demonstrate that both insect endogenous mechanisms and the microbiota can contribute to the detoxification of plant defense compounds and together they can better account for the fate of ingested plant metabolites.

Glucosinolate Abundance and Composition in Brassicaceae Influence Sequestration in a Specialist Flea Beetle.

The horseradish flea beetle Phyllotreta armoraciae exclusively feeds on Brassicaceae, which contain glucosinolates as characteristic defense compounds. Although glucosinolates are usually degraded by plant enzymes (myrosinases) to toxic isothiocyanates after ingestion, P. armoraciae beetles sequester glucosinolates. Between and within brassicaceous plants, the glucosinolate content and composition can differ drastically. But how do these factors influence sequestration in P. armoraciae? To address this question, we performed a five-day feeding experiment with three Arabidopsis thaliana lines that differ four-fold in glucosinolate content and the composition of aliphatic and indolic glucosinolates. We quantified the amounts of ingested, sequestered, and excreted glucosinolates, and analyzed the changes in glucosinolate levels and composition in beetles before and after feeding on Arabidopsis. P. armoraciae accumulated almost all ingested glucosinolate types. However, some glucosinolates were accumulated more efficiently than others, and selected glucosinolates were modified by the beetles. The uptake of new glucosinolates correlated with a decrease in the level of stored glucosinolates so that the total glucosinolate content remained stable at around 35 nmol/mg beetle fresh weight. Beetles excreted previously stored as well as ingested glucosinolates from Arabidopsis, which suggests that P. armoraciae regulate their endogenous glucosinolate level by excretion. The metabolic fate of ingested glucosinolates, i.e. the proportions of sequestered and excreted glucosinolates, depended on glucosinolate type, content, and composition in the food plant. Overall, P. armoraciae sequestered and excreted up to 41% and 31% of the total ingested aliphatic and indolic glucosinolates from Arabidopsis, respectively. In summary, we show that glucosinolate variability in Brassicaceae influences the composition but not the level of sequestered glucosinolates in P. armoraciae beetles.

Chemical convergence between plants and insects: biosynthetic origins and functions of common secondary metabolites.

Despite the phylogenetic distance between plants and insects, these two groups of organisms produce some secondary metabolites in common. Identical structures belonging to chemical classes such as the simple monoterpenes and sesquiterpenes, iridoid monoterpenes, cyanogenic glycosides, benzoic acid derivatives, benzoquinones and naphthoquinones are sometimes found in both plants and insects. In addition, very similar glucohydrolases involved in activating two-component defenses, such as glucosinolates and cyanogenic glycosides, occur in both plants and insects. Although this trend was first noted many years ago, researchers have long struggled to find convincing explanations for such co-occurrence. In some cases, identical compounds may be produced by plants to interfere with their function in insects. In others, plant and insect compounds may simply have parallel functions, probably in defense or attraction, and their co-occurrence is a coincidence. The biosynthetic origin of such co-occurring metabolites may be very different in insects as compared to plants. Plants and insects may have different pathways to the same metabolite, or similar sequences of intermediates, but different enzymes. Further knowledge of the ecological roles and biosynthetic pathways of secondary metabolites may shed more light on why plants and insects produce identical substances.

Novel family of terpene synthases evolved from trans-isoprenyl diphosphate synthases in a flea beetle.

Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene-producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon-intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors.

CYP76C1 (Cytochrome P450)-Mediated Linalool Metabolism and the Formation of Volatile and Soluble Linalool Oxides in Arabidopsis Flowers: A Strategy for Defense against Floral Antagonists.

The acyclic monoterpene alcohol linalool is one of the most frequently encountered volatile compounds in floral scents. Various linalool oxides are usually emitted along with linalool, some of which are cyclic, such as the furanoid lilac compounds. Recent work has revealed the coexistence of two flower-expressed linalool synthases that produce the (S)- or (R)-linalool enantiomers and the involvement of two P450 enzymes in the linalool oxidation in the flowers of Arabidopsis thaliana. Partially redundant enzymes may also contribute to floral linalool metabolism. Here, we provide evidence that CYP76C1 is a multifunctional enzyme that catalyzes a cascade of oxidation reactions and is the major linalool metabolizing oxygenase in Arabidopsis flowers. Based on the activity of the recombinant enzyme and mutant analyses, we demonstrate its prominent role in the formation of most of the linalool oxides identified in vivo, both as volatiles and soluble conjugated compounds, including 8-hydroxy, 8-oxo, and 8-COOH-linalool, as well as lilac aldehydes and alcohols. Analysis of insect behavior on CYP76C1 mutants and in response to linalool and its oxygenated derivatives demonstrates that CYP76C1-dependent modulation of linalool emission and production of linalool oxides contribute to reduced floral attraction and favor protection against visitors and pests.

Phyllotreta striolata flea beetles use host plant defense compounds to create their own glucosinolate-myrosinase system.

The ability of a specialized herbivore to overcome the chemical defense of a particular plant taxon not only makes it accessible as a food source but may also provide metabolites to be exploited for communication or chemical defense. Phyllotreta flea beetles are adapted to crucifer plants (Brassicales) that are defended by the glucosinolate-myrosinase system, the so-called "mustard-oil bomb." Tissue damage caused by insect feeding brings glucosinolates into contact with the plant enzyme myrosinase, which hydrolyzes them to form toxic compounds, such as isothiocyanates. However, we previously observed that Phyllotreta striolata beetles themselves produce volatile glucosinolate hydrolysis products. Here, we show that P. striolata adults selectively accumulate glucosinolates from their food plants to up to 1.75% of their body weight and express their own myrosinase. By combining proteomics and transcriptomics, a gene responsible for myrosinase activity in P. striolata was identified. The major substrates of the heterologously expressed myrosinase were aliphatic glucosinolates, which were hydrolyzed with at least fourfold higher efficiency than aromatic and indolic glucosinolates, and ß-O-glucosides. The identified beetle myrosinase belongs to the glycoside hydrolase family 1 and has up to 76% sequence similarity to other ß-glucosidases. Phylogenetic analyses suggest species-specific diversification of this gene family in insects and an independent evolution of the beetle myrosinase from other insect ß-glucosidases.

Gene coexpression analysis reveals complex metabolism of the monoterpene alcohol linalool in Arabidopsis flowers.

The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (-)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.

The Aggregation Pheromone of Phyllotreta striolata (Coleoptera: Chrysomelidae) Revisited.

Aggregations of the striped flea beetle Phyllotreta striolata on their crucifer host plants are mediated by volatiles emitted from feeding males. The male-specific sesquiterpene, (6R,7S)-himachala-9,11-diene (compound A), was shown previously to be physiologically and behaviorally active, but compound A was attractive only when combined with unnaturally high doses of the host plant volatile allyl isothiocyanate (AITC) in field trapping experiments. This indicated that our understanding of the chemical communication in this species is incomplete. Another male-specific sesquiterpenoid, (3S,9R,9aS)-3-hydroxy-3,5,5,9-tetramethyl-5,6,7,8,9,9a-hexahydro-1H-benzo[7]annulen-2(3H)-one (compound G), has been reported from an American P. striolata population. We confirmed the presence of compound G, and investigated its interaction with compound A and AITC in a P. striolata population in Taiwan. Compound G was attractive to Taiwanese P. striolata in laboratory bioassays, but significantly more beetles were attracted to a blend of compounds A and G. Under the same conditions, P. striolata showed no preference for the blend of A and G combined with a range of doses of AITC over the sesquiterpenoid blend alone. The sesquiterpenoid blend was tested further in field trapping experiments and attracted significantly more beetles than traps baited with compound A and ecologically relevant amounts of AITC. We conclude that A and G are components of the male-specific aggregation pheromone of P. striolata in Taiwan, and that the attractiveness of the pheromone is not reliant on the presence of AITC. Our results further indicate that the male-specific sesquiterpenoid blends differ qualitatively between the Taiwanese and American populations of P. striolata.

Pheromone Blend Analysis and Cross-Attraction among Populations of Maruca vitrata from Asia and West Africa.

The legume pod borer, Maruca vitrata, is a pantropical pest on leguminous crops. (E,E)-10,12-Hexadecadienal, (E,E)-10,12-hexadecadienol, and (E)-10-hexadecenal were described previously as sex pheromone components for this nocturnal moth. A blend of these components in a ratio of 100:5:5 attracted males in field trapping experiments in Benin, but not in Taiwan, Thailand, or Vietnam. This finding suggests geographic variation in the pheromone blend between Asian and West African populations of M. vitrata. We, therefore, determined the pheromone compositions of single pheromone glands of females from the three Asian regions and from Benin by gas chromatography-mass spectrometry. Additionally, we compared the responses of males from Taiwan and Benin to calling females and to gland extracts of females from both regions in laboratory no-choice and two-choice assays. Chemical analysis revealed the presence of (E,E)-10,12-hexadecadienal and (E,E)-10,12-hexadecadienol, as well as the absence of (E)-10-hexadecenal in all four populations. The relative amounts of the detected compounds did not vary significantly among the insect populations. The behavioral bioassays showed that Taiwanese and Beninese males were similarly attracted to females from both regions, as well as to their gland extracts. As a result, we did not find geographic variation in the sexual communication system of M. vitrata between West African and Asian insect populations.

Cuticular extracts from Acromis sparsa (Coleoptera: Cassidinae) mediate arrestment behavior of the commensal canestriniid mite Grandiella rugosita.

Astigmatid mites in the family Canestriniidae are often closely associated with tortoise leaf beetles (Chrysomelidae: Cassidinae). For example, the survival of the commensal canestriniid mite Grandiella rugosita depends on dispersal to the cassidine beetle Acromis sparsa. Here, we tested whether the beetle cuticle provides chemical cues for host recognition for G. rugosita. In two-choice assays with cuticular extracts from A. sparsa and the co-occurring, non-host cassidine Chelymorpha alternans offered simultaneously, mites clearly preferred the area treated with extract from their host. In no-choice assays, G. rugosita spent three times longer and moved three times slower on host cuticular extracts compared to non-host extracts and the solvent control. Analyses of the chemical composition of cuticular extracts from males and females of A. sparsa and C. alternans revealed complex mixtures of mainly methyl branched hydrocarbons, which clearly separated both species in a principal component analysis. We found no qualitative difference between males and females of either species, but in C. alternans quantitative differences between males and females were detected. Our results demonstrate that G. rugosita is able to discriminate between cuticular extracts from its host A. sparsa and the non-host C. alternans. The components eliciting the observed arrestment behavior remain to be determined.

A radiation of Psylliodes flea beetles on Brassicaceae is associated with the evolution of specific detoxification enzymes.

Flea beetles of the genus Psylliodes have evolved specialized interactions with plant species belonging to several distantly related families, mainly Brassicaceae, Solanaceae, and Fagaceae. This diverse host use indicates that Psylliodes flea beetles are able to cope with different chemical defense metabolites, including glucosinolates, the characteristic defense metabolites of Brassicaceae. Here we investigated the evolution of host use and the emergence of a glucosinolate-specific detoxification mechanism in Psylliodes flea beetles. In phylogenetic analyses, Psylliodes species clustered into four major clades, three of which contained mainly species specialized on either Brassicaceae, Solanaceae, or Fagaceae. Most members of the fourth clade have broader host use, including Brassicaceae and Poaceae as major host plant families. Ancestral state reconstructions suggest that Psylliodes flea beetles were initially associated with Brassicaceae and then either shifted to Solanaceae or Fagaceae, or expanded their host repertoire to Poaceae. Despite a putative ancestral association with Brassicaceae, we found evidence that the evolution of glucosinolate-specific detoxification enzymes coincides with the radiation of Psylliodes on Brassicaceae, suggesting that these are not required for using Brassicaceae as hosts but could improve the efficiency of host use by specialized Psylliodes species.

Different myrosinases activate sequestered glucosinolates in larvae and adults of the horseradish flea beetle.

ß-Glucosidases play an important role in the chemical defense of many insects by hydrolyzing and thereby activating glucosylated pro-toxins that are either synthesized de novo or sequestered from the insect's diet. The horseradish flea beetle, Phyllotreta armoraciae, sequesters pro-toxic glucosinolates from its brassicaceous host plants and possesses endogenous ß-thioglucosidase enzymes, known as myrosinases, for glucosinolate activation. Here, we identify three myrosinase genes in P. armoraciae (PaMyr) with distinct expression patterns during beetle ontogeny. By using RNA interference, we demonstrate that PaMyr1 is responsible for myrosinase activity in adults, whereas PaMyr2 is responsible for myrosinase activity in larvae. Compared to PaMyr1 and PaMyr2, PaMyr3 was only weakly expressed in our laboratory population, but may contribute to myrosinase activity in larvae. Silencing of PaMyr2 resulted in lower larval survival in a predation experiment and also reduced the breakdown of sequestered glucosinolates in uninjured larvae. This suggests that PaMyr2 is involved in both activated defense and the endogenous turnover of sequestered glucosinolates in P. armoraciae larvae. In activity assays with recombinant enzymes, PaMyr1 and PaMyr2 preferred different glucosinolates as substrates, which was consistent with the enzyme activities in crude protein extracts from adults and larvae, respectively. These differences were unexpected because larvae and adults sequester the same glucosinolates. Possible reasons for different myrosinase activities in Phyllotreta larvae and adults are discussed.

Rapid and Selective Absorption of Plant Defense Compounds From the Gut of a Sequestering Insect.

Many herbivorous insects exploit defense compounds produced by their host plants for protection against predators. Ingested plant defense compounds are absorbed via the gut epithelium and stored in the body, a physiological process that is currently not well understood. Here, we investigated the absorption of plant defense compounds from the gut in the horseradish flea beetle, Phyllotreta armoraciae, a specialist herbivore known to selectively sequester glucosinolates from its brassicaceous host plants. Feeding experiments using a mixture of glucosinolates and other glucosides not found in the host plants showed a rapid and selective uptake of glucosinolates in adult beetles. In addition, we provide evidence that this uptake mainly takes place in the foregut, whereas the endodermal midgut is the normal region of absorption. Absorption via the foregut epithelium is surprising as the apical membrane is covered by a chitinous intima. However, we could show that this cuticular layer differs in its structure and overall thickness between P. armoraciae and a non-sequestering leaf beetle. In P. armoraciae, we observed a thinner cuticle with a less dense chitinous matrix, which might facilitate glucosinolate absorption. Our results show that a selective and rapid uptake of glucosinolates from the anterior region of the gut contributes to the selective sequestration of glucosinolates in P. armoraciae.

Metabolization and sequestration of plant specialized metabolites in insect herbivores: Current and emerging approaches.

Herbivorous insects encounter diverse plant specialized metabolites (PSMs) in their diet, that have deterrent, anti-nutritional, or toxic properties. Understanding how they cope with PSMs is crucial to understand their biology, population dynamics, and evolution. This review summarizes current and emerging cutting-edge methods that can be used to characterize the metabolic fate of PSMs, from ingestion to excretion or sequestration. It further emphasizes a workflow that enables not only to study PSM metabolism at different scales, but also to tackle and validate the genetic and biochemical mechanisms involved in PSM resistance by herbivores. This review thus aims at facilitating research on PSM-mediated plant-herbivore interactions.

Male Phyllotreta striolata (F.) produce an aggregation pheromone: identification of male-specific compounds and interaction with host plant volatiles.

The chrysomelid beetle Phyllotreta striolata is an important pest of Brassicaceae in Southeast Asia and North America. Here, we identified the aggregation pheromone of a population of P. striolata from Taiwan, and host plant volatiles that interact with the pheromone. Volatiles emitted by feeding male P. striolata attracted males and females in the field. Headspace volatile analyses revealed that six sesquiterpenes were emitted specifically by feeding males. Only one of these, however, elicited an electrophysiological response from antennae of both sexes. A number of host plant volatiles, e.g., 1-hexanol, (Z)-3-hexen-1-ol, and the glucosinolate hydrolysis products allyl isothiocyanate (AITC), 3-butenyl isothiocyanate, and 4-pentenyl isothiocyanate also elicited clear responses from the antenna. The active male-specific compound was identified as (+)-(6R,7S)-himachala-9,11-diene by chiral stationary phase gas-chromatography with coupled mass spectrometry, and by comparison with reference samples from Abies nordmanniana, which is known to produce the corresponding enantiomer. The pheromone compound was synthesized starting from (-)-α-himachalene isolated from Cedrus atlantica. Under field conditions, the activity of the synthetic pheromone required concomitant presence of the host plant volatile allyl isothiocyanate. However, both synthetic (+)-(6R,7S)-himachala-9,11-diene alone and in combination with AITC were attractive in a two-choice laboratory assay devoid of other natural olfactory stimuli. We hypothesize that P. striolata adults respond to the pheromone only if specific host volatiles are present. In the same laboratory set up, more beetles were attracted by feeding males than by the synthetic stimuli. Thus, further research will be necessary to reveal the components of a more complex blend of host or male-produced semiochemicals that might enhance trap attractiveness in the field.