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.

Production, identification, and field evaluation of sex pheromone from calling females in Diaphania angustalis (Lepidoptera: Crambidae).

Insect sex pheromones play a crucial role in the mate finding and calling behavior of Lepidoptera pests. Currently, little is known about the chemical ecology of Diaphania angustalis Snellen (Lepidoptera: Crambidae), a severe and important defoliator attacking the medicinal plant, Alstonia scholaris. In the present study, the pheromone components of D. angustalis females were investigated using electrophysiological and behavioral methods. Distilled hexane extracts of female pheromone glands were analyzed through electroantennogram (EAG) and gas chromatography-electroantennogram detector (GC-EAD), and the active compounds were identified through gas chromatography-mass spectrometry (GC-MS). Production peak of female sex pheromone occurred on the third day of age at 5 h into the scotophase with the EAG test, and the hexane extracts were attractive to males in the wind tunnel test. GC-EAD analysis of virgin males to gland extracts that were subsequently evaluated showed two active compounds, (E,E)-10,12-hexadecadienal (E10E12-16:Ald) and (E,E)-10,12-hexadecadien-1-ol (E10E12-16:OH), based on comparison of retention time and mass spectrum, with suitable synthetic compounds. Under laboratory conditions, the blend of E10E12-16:Ald and E10E12-16:OH in a ratio of 9:1 elicited a stronger EAG response than other treatments or a single component. In the field, more male moths were captured by traps baited with the mixture of E10E2-16:Ald and E10E2-16:OH in a ratio of 9:1, whereas a mixture of 8:1 and 10:1 also caught males. Accordingly, E10E2-16:Ald and E10E2-16:OH were regarded as the major sex pheromone components in D. angustalis females.

Sexual Deception in the Eucera-Pollinated Ophrys leochroma: A Chemical Intermediate between Wasp- and Andrena-Pollinated Species.

Ophrys flowers mimic sex pheromones of attractive females of their pollinators and attract males, which attempt to copulate with the flower and thereby pollinate it. Virgin females and orchid flowers are known to use the same chemical compounds in order to attract males. The composition of the sex pheromone and its floral analogue, however, vary between pollinator genera. Wasp-pollinated Ophrys species attract their pollinators by using polar hydroxy acids, whereas Andrena-pollinated species use a mixture of non-polar hydrocarbons. The phylogeny of Ophrys shows that its evolution was marked by episodes of rapid diversification coinciding with shifts to different pollinator groups: from wasps to Eucera and consequently to Andrena and other bees. To gain further insights, we studied pollinator attraction in O. leochroma in the context of intra- and inter-generic pollinator shifts, radiation, and diversification in the genus Ophrys. Our model species, O. leochroma, is pollinated by Eucera kullenbergi males and lies in the phylogeny between the wasp and Andrena-pollinated species; therefore, it is a remarkable point to understand pollinator shifts. We collected surface extracts of attractive E. kullenbergi females and labellum extracts of O. leochroma and analyzed them by using gas chromatography with electroantennographic detection (GC-EAD) and gas chromatography coupled with mass spectrometry (GC-MS). We also performed field bioassays. Our results show that O. leochroma mimics the sex pheromone of its pollinator's female by using aldehydes, alcohols, fatty acids, and non-polar compounds (hydrocarbons). Therefore, in terms of the chemistry of pollinator attraction, Eucera-pollinated Ophrys species might represent an intermediate stage between wasp- and Andrena-pollinated orchid species.

Synthesis of (6R,12R)-6,12-Dimethylpentadecan-2-one, the Female-Produced Sex Pheromone from Banded Cucumber Beetle Diabrotica balteata, Based on a Chiron Approach.

Herein we describe a synthesis of (6R,12R)-6,12-dimethylpentadecan-2-one (5), the female produced sex pheromone of banded cucumber beetle Diabrotica balteata Le Conte, from (R)-4-methyl-5-valerolactone, a methyl-branched chiron.

Discovery of pyrazines as pollinator sex pheromones and orchid semiochemicals: implications for the evolution of sexual deception.

Sexually deceptive orchids employ floral volatiles to sexually lure their specific pollinators. How and why this pollination system has evolved independently on multiple continents remains unknown, although preadaptation is considered to have been important. Understanding the chemistry of sexual deception is a crucial first step towards solving this mystery. The combination of gas chromatography-electroantennographic detection (GC-EAD), GC-MS, synthesis and field bioassays allowed us to identify the volatiles involved in the interaction between the orchid Drakaea glyptodon and its sexually attracted male thynnine wasp pollinator, Zaspilothynnus trilobatus. Three alkylpyrazines and one novel hydroxymethyl pyrazine were identified as the sex pheromone of Z. trilobatus and are also used by D. glyptodon for pollinator attraction. Given that our findings revealed a new chemical system for plants, we surveyed widely across representative orchid taxa for the presence of these compounds. With one exception, our chemical survey failed to detect pyrazines in related genera. Collectively, no evidence for preadaptation was found. The chemistry of sexual deception is more diverse than previously known. Our results suggest that evolutionary novelty may have played a key role in the evolution of sexual deception and highlight the value of investigating unusual pollination systems for advancing our understanding of the role of chemistry in evolution.

Photoremovable protecting groups as controlled-release device for sex pheromone.

Photoremovable protecting groups (PRPGs) were demonstrated as a delivery device for controlled release of pheromone under both UV light (≥350 nm) and direct sunlight irradiation. In the present work, (Z)-11-hexadecen-1-ol (sex pheromone of Chilo infuscatellus Snellen) was chemically caged by four different photoremovable protecting groups (7-hydroxy-4-hydroxymethylcoumarin, 1-pyrenemethanol, 9-anthracenemethanol and 2-(hydroxymethyl)anthraquinone) individually. Photophysical studies showed that the caged pheromone with coumarin, pyrene and anthracene derivatives exhibited strong fluorescence. Controlled release of (Z)-11-hexadecen-1-ol was achieved by irradiating the caged compounds in aqueous ethanol both under UV and sunlight. Further, to mimic the environmental conditions, controlled release of (Z)-11-hexadecen-1-ol was also studied in soil medium under direct sunlight. Thermogravimetric analysis showed that caging of (Z)-11-hexadecen-1-ol by PRPGs significantly reduced its volatility. Bioassay experiments indicated that PRPGs are harmless to soil bacteria (Azotobacter sp. and Pseudomonas aeruginosa) and in vitro cytotoxicity studies on eukaryotic L929 cells showed that PRPGs are also non-toxic. Field bioassays were performed using caged pheromone against maize stalk borer (Chilo partellus) and the results showed that the caged alcohol is effective in a number of moths catches instead of free alcohol in a blend for a longer period of time. Our studies indicated that use of PRPGs as delivery device for controlled release of pheromone by sunlight holds great interest for field applications.

Terminal fatty-acyl-CoA desaturase involved in sex pheromone biosynthesis in the winter moth (Operophtera brumata).

The winter moth (Operophtera brumata L., Lepidoptera: Geometridae) utilizes a single hydrocarbon, 1,Z3,Z6,Z9-nonadecatetraene, as its sex pheromone. We tested the hypothesis that a fatty acid precursor, Z11,Z14,Z17,19-nonadecanoic acid, is biosynthesized from α-linolenic acid, through chain elongation by one 2-carbon unit, and subsequent methyl-terminus desaturation. Our results show that labeled α-linolenic acid is indeed incorporated into the pheromone component in vivo. A fatty-acyl-CoA desaturase gene that we found to be expressed in the abdominal epidermal tissue, the presumed site of biosynthesis for type II pheromones, was characterized and expressed heterologously in a yeast system. The transgenic yeast expressing this insect derived gene could convert Z11,Z14,Z17-eicosatrienoic acid into Z11,Z14,Z17,19-eicosatetraenoic acid. These results provide evidence that a terminal desaturation step is involved in the winter moth pheromone biosynthesis, prior to the decarboxylation.

Attract-and-kill: a new strategy for the management of the potato tuber moths Phthorimaea operculella (Zeller) and Symmetrischema tangolias (Gyen) in potato: laboratory experiments towards optimising pheromone and insecticide concentration.

BACKGROUND: Sex pheromones of the potato tuber moths Phthorimaea operculella (Zeller) and Symmetrischema tangolias (Gyen) are ideal tools to monitor pest flight activity but are not used as means of control. The aim of the present study was to test the suitability of an attract-and-kill strategy consisting of pure pheromones and the contact insecticide cyfluthrin as the active ingredient, formulated with plant oils and ultraviolet absorbers, and applied in droplet sizes of 100 microL. RESULTS: Cyfluthrin at a concentration of 5 g L(-1) resulted in the highest and fastest killing of males after 48 h, with a 100% mortality after 3-4 days. In contrast, control males survived for 13 days. In olfactometer experiments, the pheromone concentration of 0.5 g L(-1) was significantly most attractive against eight virgin females. At controlled conditions (20 degrees C), no reduction in efficacy of the attract-and-kill formulation was observed for a minimum period of 36 days, whereas under natural environmental conditions the efficacy reduced gradually after day 6 of exposure. The longer the droplet was exposed, the longer was the time to reach 100% mortality of males. CONCLUSIONS: Compared with attract-and-kill studies for other pest species, the results are promising as a means of achieving highly effective control of potato tuber moths under field conditions.

Ménage à trois-two endemic species of deceptive orchids and one pollinator species.

In the sexually deceptive orchid genus Ophrys, reproductive isolation is based on the specific attraction of males of a single pollinator species by mimicking the female species-specific sex pheromone. Changes in the odor composition can lead to hybridization and speciation by the attraction of a new pollinator that acts as an isolation barrier toward other sympatrically occurring Ophrys species. On Sardinia, we investigated the evolutionary origin of two sympatrically occurring endemic species, Ophrys chestermanii and O. normanii, which are both pollinated by males of the cuckoo bumblebee Bombus vestalis. Chemical and electrophysiological analyses of floral scent and genetic analyses with amplified fragment length polymorphisms and plastid-markers clearly showed that O. normanii is neither a hybrid nor a hybrid species. The two species evolved from different ancestors, viz. O. normanii from O. tenthredinifera and O. chestermanii from O. annae, and converged to the same pollinator attracted by the same bouquet of polar compounds. In spite of sympatry, pollinator sharing and overlapping blooming periods, no evidence has been obtained for gene flow between O. chestermanii and O. normanii indicating an unusual case among sexually deceptive orchids in which postmating rather than premating reproductive isolation mechanisms strongly prevent interspecific gene flow.

Improved synthesis of (3E,7Z)-3,7-tetradecadienyl acetate, the major sex pheromone constituent of the potato pest Symmetrischema tangolias (Gyen).

An efficient six-step synthesis of (3E,7Z)-3,7-tetradecadienyl acetate, the major component of the sex pheromone of the potato pest Symmetrischema tangolias (Gyen), is described, starting from the commercially available dihydropyran. The stereoselective formation of the 7Z double bond is accomplished by a Wittig reaction, while the 3E double bond is formed by a modified Knoevenagel condensation. The overall yield of the synthesis is 28%, giving the final product in high stereochemical purity (95%). The simplicity and the low cost of the herein reported synthesis suggest the potential practical use of the above pheromone in integrated management programs, for this serious insect pest.

Vitamin D supplementation increases the attractiveness of males' scent for female Iberian rock lizards.

Evolutionary theory proposes that signals used in sexual selection can only be stable if they are honest and condition dependent. However, despite the fact that chemical signals are used by many animals, empirical research has mainly focused on visual and acoustic signals. Vitamin D is an essential nutrient for lizards, but in some lizards its precursor (cholesta-5,7-dien-3-ol=provitamin D) is found in femoral gland secretions, which males use for scent marking and intraspecific communication. By allocating provitamin D to secretions, males might need to divert vitamin D from metabolism. This might be costly and condition dependent. We tested whether diet quality affected chemical signals of male Iberian rock lizards (Lacerta monticola) and its consequences for sexual selection. After experimental supplementation of dietary vitamin D, males increased the proportion of provitamin D in femoral secretions. Further experiments showed that females detected these changes in males' signals by chemosensory cues, and discriminated provitamin D, and changes in its concentration, from similar steroids (i.e. cholesterol) found in secretions. Moreover, females preferred areas scent marked by males with more provitamin D in their secretions. This mechanism would confer honesty to chemical signals of male lizards, and, thus, females may rely on it to select high-quality males. We suggest that the allocation of vitamins and other essential nutrients to either visual (e.g. carotenoids) or chemical ornaments might be the common basis of honest sexual displays in many animals.

The effect of pollination on floral fragrance in thistles.

We investigated postpollination changes in fragrance composition and emission rates, as well as pollinator discrimination in hand-pollinated flower heads of two thistle species: Canada thistle (Cirsium arvense) and sandhill thistle (C. repandum). Following pollination, neither species emitted any novel compounds that could function as repellents. Scent emission rates declined in pollinated plants of both species by approximately 89% within 48 hr. This decline was evident in all 13 scent components of C. arvense. Apis mellifera, the dominant pollinator in the study population of C. arvense, was nearly three times more likely to visit an unpollinated rather than a pollinated flower head. A more complex pattern was observed for C. repandum, whose scent comprised 42 compounds. Quantities of aromatic and sesquiterpenoid volatiles declined after pollination, whereas two classes of scent compounds, fatty acid derivatives and monoterperpenoids, continued to be emitted. In C. repandum, discrimination against pollinated flower heads by Papilio palamedes (its primary pollinator) was not as marked. Unpollinated control plants of both species maintained moderate levels of scent production throughout this experiment, demonstrating that senescence and floral advertisement may be delayed until pollination has occurred. We expect postpollination changes in floral scent contribute to communication between plants with generalized pollinator spectra and their floral visitors. This study provides the first field study of such a phenomenon outside of orchids.

Identification of two sex pheromone components of the potato aphid, Macrosiphum euphorbiae (Thomas).

Females of the potato aphid Macrosiphum euphorbiae exhibit typical calling behavior, with virgin female oviparae raising their back legs off the substrate to release sex pheromone from glands on the tibia. Airborne collections from calling oviparae were analyzed by GC and GC-MS to determine if, like the majority of aphids examined to date, they produced (1R,4aS,7S,7aR)-nepetalactol (1) and (4aS,7S,7aR)-nepetalactone (2). Both components were present and produced in ratios that varied with age from 4:1 to 2:1. The relative stereochemical configurations of these components were determined by GC-coinjection of the aphid-derived sample with synthetic standards on both HP-1 and DB-Wax GC columns. The absolute stereochemical configuration of the nepetalactol (determined from approximately 15 microg of material in an air entrainment sample) was determined as (1R,4aS,7S,7aR)-1 by derivatization of the aphid sample with (S)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride (Mosher's acid chloride) to generate a diastereoisomer that was compared to synthetic samples by NMR spectroscopy and GC. In bioassays in the wind tunnel, M. euphorbiae males responded to potato plants with oviparae but not to unattacked plants or those infested with parthenogenetically reproducing apterae. In no-choice laboratory bioassays, the same level of male response was observed to virgins and to the 3:1-5:1 synthetic blends of nepetalactol (1):nepetalactone (2). However, the time taken to reach the source was significantly less to virgin females than to the synthetic pheromone blends. In all cases, males walked rather than flew to the source. Males showed lower responses to a 1:1 synthetic mixture and did not respond to either of the components when presented alone. Under field conditions, few M. euphorbiae males were captured in traps baited with different ratios of the synthetic pheromone. Possible reasons for the different responses under laboratory and field conditions are discussed.

Host-plant specialization in pheromone strains of the European corn borer Ostrinia nubilalis in France.

European corn borer (ECB) feeding on maize (Zea mais), mugwort (Artemisia vulgaris), and hop (Humulus lupulus) are genetically different in France and referred to as host-plant races. Here, we investigated sex pheromone composition as a possible trait linked to the host plant. ECB host races were sampled from 13 different sites in France. GC-MS analysis of female pheromone showed that 175 out of 176 maize females belonged to the Z type with one hybrid. In contrast, mugwort and hop females belonged almost exclusively to the E type. No Z females were found on these plants and only 2 females out of 169 were hybrids. In the three sites of sympatry, the hybrid proportion was far from Hardy-Weinberg expectations. Wind tunnel experiments showed that 76-79% of maize males from three populations were attracted by Z females, whereas neither mugwort nor hop males were. Mugwort males from Toussus-le-Noble were attracted by E females originating from an American maize strain. These data showed that maize, mugwort, and hop host races of O. nubilalis differ not only in their host plant but also in the sex pheromone they use. Because mugwort and hop are putative ancestral host plants, these results are discussed from the point of view of evolutionary scenarios for the emergence of Z and E strains.

The chemistry of sexual deception in an orchid-wasp pollination system.

The "sexually deceptive" orchid Chiloglottis trapeziformis attracts males of its pollinator species, the thynnine wasp Neozeleboria cryptoides, by emitting a unique volatile compound, 2-ethyl-5-propylcyclohexan-1,3-dione, which is also produced by female wasps as a male-attracting sex pheromone.

Genetic isolation between two sympatric host-plant races of the European corn borer, Ostrinia nubilalis Hübner. I. Sex pheromone, moth emergence timing, and parasitism.

Adaptation to different environments may be a powerful source of genetic differentiation between populations. The biological traits selected in each environment can pleiotropically induce assortative mating between individuals of these genetically differentiated populations. This situation may facilitate sympatric speciation. Successful host shifts in phytophagous insects provide some of the best evidence for the ecological speciation that occurs, or has occurred, in sympatry. The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), colonized maize after its introduction into Europe by humans about 500 years ago. In northern France, two sympatric host races feed on maize (Zea mays) and mugwort (Artemisia vulgaris), respectively. We investigated the factors involved in the genetic isolation of these two races at a field site near Paris, France. We identified two biological differences that might make a significant contribution to the genetic divergence between sympatric populations feeding on the two host plants. First, assortative mating may be due to differences in the moth emergence pattern between the two races: mugwort-race moths emerged on average 10 days earlier than maize-race moths. In addition, the males emerged earlier than females in both races. Hence, the likelihood of mating between maize-race males and mugwort-race females was higher than that of mating between mugwort-race males and maize-race females. Second, the females feeding on mugwort and maize produced sex pheromones with different E/Z isomeric ratios of delta-11-tetradecenyl acetate. This difference in mate recognition systems reinforces the potential for assortative mating in the two races. During the experiment, overwintering mortality was much lower on maize than on mugwort. This difference was due to a braconid parasitoid wasp, Macrocentrus cingulum, that killed more than 50% of the larvae overwintering on mugwort but did not infest larvae diapausing on maize. Hence, by colonizing maize, European corn borer populations probably escaped from numerous predators, competitors, and parasitoids, such as M. cingulum. This decrease in host-associated selection may have favored the colonization of this new host. Finally, throughout this experiment we observed selection at two allozyme loci (or at linked loci): Tpi and Mpi. The Tpi locus is tightly linked with the genes involved in the response of the male to the sex pheromone and in developmental timing. The location of these traits on the Z chromosome may play a role in shortening the time required for the evolution of premating barriers.

Sex pheromone mimicry in the early spider orchid (ophrys sphegodes): patterns of hydrocarbons as the key mechanism for pollination by sexual deception.

We investigated the female-produced sex pheromone of the solitary bee Andrena nigroaenea and compared it with floral scent of the sexually deceptive orchid Ophrys sphegodes which is pollinated by Andrena nigroaenea males. We identified physiologically and behaviorally active compounds by gas chromatography with electroantennographic detection, gas chromatography-mass spectrometry, and behavioral tests in the field. Dummies scented with cuticle extracts of virgin females or of O. sphegodes labellum extracts elicited significantly more male reactions than odorless dummies. Therefore, copulation behavior eliciting semiochemicals are located on the surface of the females' cuticle and the surface of the flowers. Within bee and orchid samples, n-alkanes and n-alkenes, aldehydes, esters, all-trans-farnesol and all-trans-farnesyl hexanoate triggered electroantennographic responses in male antennae. Most of the alkanes and alkenes occurred in similar patterns both in the bees and orchids. O. sphegodes leaf extracts contained mostly the same compounds but in different proportions. In behavioral tests with synthetic compounds, blends of alkenes triggered significantly more approaches and pounces of the males whereas alkanes were not more attractive than odorless dummies. Since alkanes and alkenes together were most attractive, we conclude they constitute the bees' sex pheromone as well as the pseudocopulation-behavior releasing orchid-odor bouquet.