The effect of developmental temperature on olfaction in a moth revealed by its interaction with body mass.

There is a growing interest in the effects of climate warming on olfaction, as temperature may affect this essential sense. In insects, the response of the olfactory system to developmental temperature might be mediated by body size or mass because body size and mass are negatively affected by developmental temperature in most ectotherms. We tested this hypothesis of a mass-mediated effect of developmental temperature on olfaction in the moth Spodoptera littoralis. We measured the olfactory sensitivity of male to female sex pheromone and five plant odors using electroantennography. We compared males reared at an optimal temperature (25 °C with a daily fluctuation of ±5 °C) and at a high temperature (33 ± 5 °C) close to the upper limit of S. littoralis. On average, the olfactory sensitivity of males did not differ between the two developmental temperatures. However, our analyses revealed an interaction between the effects of developmental temperature and body mass on the detection of the six chemicals tested. This interaction is explained by a positive relationship between antennal sensitivity and body mass observed only with the high developmental temperature. Our results show that the effect of developmental temperature may not be detected when organism size is ignored.

Functional Characterization of Odorant Receptors for Sex Pheromone (Z)-11-Hexadecenol in Orthaga achatina.

Pheromone receptor (PR)-mediated transduction of sex pheromones to electrophysiological signals is the basis for sex pheromone communication. Orthaga achatina, a serious pest of the camphor tree, uses a mixture of four components (Z11-16:OAc, Z11-16:OH, Z11-16:Ald, and Z3,Z6,Z9,Z12,Z15-23:H) as its sex pheromone. In this study, we identified five PR genes (OachPR1-5) by phylogenetic analysis. Further RT-PCR and qPCR experiments showed that PR1-3 were specifically expressed in male antennae, while PR4 was significantly female-biased in expression. Functional characterization using the XOE-TEVC assay demonstrated that PR1 and PR3 both responded strongly to Z11-16:OH, while PR1 and PR3 had a weak response to Z3,Z6,Z9,Z12,Z15-23:H and Z11-16:Ald, respectively. Finally, two key amino acid residues (N78 and R331) were confirmed to be essential for binding of PR3 with Z11-16:OH by molecular docking and site-directed mutagenesis. This study helps understand the sex pheromone recognition molecular mechanism of O. achatina.

The Asymmetric Total Synthesis of the Female-Produced Sex Pheromone of the Tea Tussock Moth.

The tea tussock moth is a pest that damages tea leaves, affecting the quality and yield of tea and causing huge economic losses. The efficient asymmetric total synthesis of the sex pheromone of the tea tussock moth was achieved using commercially available starting materials with a 25% overall yield in 11 steps. Moreover, the chiral moiety was introduced by Evans' template and the key C-C bond construction was accomplished through Julia-Kocienski olefination coupling. The synthetic sex pheromone of the tea tussock moth will facilitate the subsequent assessment and implementation of pheromones as environmentally friendly tools for pest management.

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans.

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males. This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

Functional Characterization of Peripheral Neurons and a Receptor Recognizing Sex Pheromones in Hyphantria cunea (Erebidae).

Hyphantria cunea (Lepidoptera: Erebidae) is difficult and costly to control as a quarantine pest found globally. Sex pheromone trapping is an effective measure for its population monitoring and control; however, the peripheral neural mechanism of sex pheromone recognition in H. cunea remains unclear. An electrophysiological analysis showed that both male and female moths of H. cunea responded to four components of sex pheromones and the responses of male moths were stronger than those of the female moths. We identified three types of trichoid sensilla (ST) responsive to sex pheromones using the single sensillum recording technique. Each type was involved in recognizing 9R, 10S-epoxy-1, Z3, Z6-heneicosatriene (1, Z3, Z6-9S, 10R-epoxy-21Hy). Four peripheral neurons involved in the olfactory encoding of sex pheromones were identified. Four candidate pheromone receptor (PR) genes, HcunPR1a, HcunPR1b, HcunPR3, and HcunPR4, were screened by transcriptome sequencing. All of them were highly expressed in the antennae of males, except for HcunPR4, which was highly expressed in the antennae of females. Functional identification showed that HcunPR1a responded to sex pheromone. Other HcunPRs were not functionally identified. In summary, neurons involved in sex pheromone recognition of H. cunea were located in the ST, and HcunPR1a recognized secondary pheromone components 1, Z3, Z6-9S, 10R-epoxy-21Hy. Interestingly, PRs that recognize the main components of the sex pheromone may be located in an unknown branch of the olfactory receptor and merit further study. Our findings provide a better understanding of the peripheral neural coding mechanism of type II sex pheromones, and HcunPR1a may provide a target for the subsequent development of highly effective and specific biopesticides for H. cunea.

Functional characterization of sex pheromone receptors PflaOR29 and PflaOR44 involved in the chemoreception of a diurnal moth, Phauda flammans (Walker) (Lepidoptera: Phaudidae).

Recognition of sex pheromones released by heterosexual moths via sex pheromone receptors is key for establishing mating connections in moths. The day-flying moth Phauda flammans is an oligophagous pest in southern cities of China and Southeast Asian countries. Our previous study reported that male P. flammans can be attracted to two sex pheromone compounds [Z-9-hexadecenal and (Z, Z, Z)-9,12,15-octadecadienal] released by females in the field; however, the mechanism of olfactory recognition is not clear. In this study, two sex pheromone receptor genes (PflaOR29 and PflaOR44) were cloned. Among the different tissues, both PflaOR29 and PflaOR44 were highly expressed in the antennae of mated male adults. At different developmental stages, the expression levels of PflaOR29 and PflaOR44 were significantly greater in mated male adults than other stages. The fluorescence signals of PflaOR29 and PflaOR44 were mostly distributed on the dorsal side of the antennae, with a large number of trichoid sensilla. The results of the gene function of PflaOR29 and PflaOR44 based on a Drosophila empty neuron heterologous expression system indicated that PflaOR29 strongly responded to (Z, Z, Z)-9,12,15-octadecadienal but not to Z-9-hexadecenal, whereas PflaOR44 did not respond to the two sex pheromones. Our findings clarify the sex pheromone receptor gene corresponding to (Z, Z, Z)-9,12,15-octadecatrienal. These results provide essential information for analyzing the mechanism of sexual communication in diurnal moths and for identifying target genes for the development of efficient attractants.

Total Synthesis of a Peptide Diatom Sex Pheromone Bearing a Sulfated Aspartic Acid.

The peptide sex-inducing pheromone SIP+ (1) bearing an unusual sulfated aspartic acid residue induces sexual reproduction in diatom populations. Herein, we report the first total synthesis of SIP+ using both a sulfated building block approach and a solid-phase peptide synthesis (SPPS)-compatible late-stage sulfation strategy to assemble the natural product. The modular approaches provide concise routes to useful quantities of the natural product for future structure activity relationship studies examining the role of SIP+ in diatom biology.

Exploration-exploitation model of moth-inspired olfactory navigation.

Navigation of male moths towards females during the mating search offers a unique perspective on the exploration-exploitation (EE) model in decision-making. This study uses the EE model to explain male moth pheromone-driven flight paths. Wind tunnel measurements and three-dimensional tracking using infrared cameras have been leveraged to gain insights into male moth behaviour. During the experiments in the wind tunnel, disturbance to the airflow has been added and the effect of increased fluctuations on moth flights has been analysed, in the context of the proposed EE model. The exploration and exploitation phases are separated using a genetic algorithm to the experimentally obtained dataset of moth three-dimensional trajectories. First, the exploration-to-exploitation rate (EER) increases with distance from the source of the female pheromone is demonstrated, which can be explained in the context of the EE model. Furthermore, our findings reveal a compelling relationship between EER and increased flow fluctuations near the pheromone source. Using an olfactory navigation simulation and our moth-inspired navigation model, the phenomenon where male moths exhibit an enhanced EER as turbulence levels increase is explained. This research extends our understanding of optimal navigation strategies based on general biological EE models and supports the development of bioinspired navigation algorithms.

A ghost moth olfactory prototype of the lepidopteran sex communication.

Sex role differentiation is a widespread phenomenon. Sex pheromones are often associated with sex roles and convey sex-specific information. In Lepidoptera, females release sex pheromones to attract males, which evolve sophisticated olfactory structures to relay pheromone signals. However, in some primitive moths, sex role differentiation becomes diverged. Here, we introduce the chromosome-level genome assembly from ancestral Himalaya ghost moths, revealing a unique olfactory evolution pattern and sex role parity among Lepidoptera. These olfactory structures of the ghost moths are characterized by a dense population of trichoid sensilla, both larger male and female antennal entry parts of brains, compared to the evolutionary later Lepidoptera. Furthermore, a unique tandem of 34 odorant receptor 19 homologs in Thitarodes xiaojinensis (TxiaOr19) has been identified, which presents overlapped motifs with pheromone receptors (PRs). Interestingly, the expanded TxiaOr19 was predicted to have unconventional tuning patterns compared to canonical PRs, with nonsexual dimorphic olfactory neuropils discovered, which contributes to the observed equal sex roles in Thitarodes adults. Additionally, transposable element activity bursts have provided traceable loci landscapes where parallel diversifications occurred between TxiaOr19 and PRs, indicating that the Or19 homolog expansions were diversified to PRs during evolution and thus established the classic sex roles in higher moths. This study elucidates an olfactory prototype of intermediate sex communication from Himalaya ghost moths.

The genetic basis of incipient sexual isolation in Drosophila melanogaster.

Speciation is a fundamental evolutionary process but the genetic changes accompanying speciation are difficult to determine since true species do not produce viable and fertile offspring. Partially reproductively isolated incipient species are useful for assessing genetic changes that occur prior to speciation. Drosophila melanogaster from Zimbabwe, Africa are partially sexually isolated from other D. melanogaster populations whose males have poor mating success with Zimbabwe females. We used the North American D. melanogaster Genetic Reference Panel (DGRP) to show that there is significant genetic variation in mating success of DGRP males with Zimbabwe females, to map genetic variants and genes associated with variation in mating success and to determine whether mating success to Zimbabwe females is associated with other quantitative traits previously measured in the DGRP. Incipient sexual isolation is highly polygenic and associated with the common African inversion In(3R)K and the amount of the sex pheromone 5,9-heptacosadiene in DGRP females. We functionally validated the effect of eight candidate genes using RNA interference to provide testable hypotheses for future studies investigating the molecular genetic basis of incipient sexual isolation in D. melanogaster.

Transcriptome analysis reveals the pheromone synthesis mechanism and mating response in Monochamus saltuarius (Coleoptera, Cerambycidae).

The pinewood nematode Bursaphelenchus xylophilus (Steiner and Buhrer 1934) causes pine wilt disease, which severely affects the biodiversity and economy of Eurasian coniferous forests. Monochamus saltuarius (Coleoptera, Cerambycidae) was first identified as nematode vectors in Liaoning Province, China, in 2017. M. saltuarius has high mating efficiency and reproductive capabilities, pheromones are crucial in these processes. However, the mechanisms of pheromone synthesis in M. saltuarius are unclear. This study performed morphometric and transcriptomic analyses of the internal reproductive systems of males and females at different developmental stages and analyzed mate selection behavior. We found a significant difference in the morphology of internal reproductive systems between sexually immature and mature insects. A total of 58 and 64 pheromone biosynthesis genes were identified in females and males, respectively. The expression of the analyzed genes differed between males and females in the initial and subsequent synthesis processes. Interference experiment indicated that knocking down SDR1 gene in male M. saltuarius reduces the content of pheromones. Behavioral analyses found that males preferred virgin females. This study identified key pheromone genes and synthesis pathway that could serve as potential targets for disrupting mating in M. saltuarius through the development of novel biological agents using genetic engineering techniques.

Dibutyl phthalate released by solitary female locusts mediates sexual communication at low density.

Sex pheromones play a crucial role in mate location and reproductive success. Insects face challenges in finding mates in low-density environments. The population dynamics of locusts vary greatly, ranging from solitary individuals to high-density swarms, leading to multiple-trait divergence between solitary and gregarious phases. However, differences in sexual communication between solitary and gregarious locusts have not been sufficiently explored. Herein, we found that solitary locusts but not gregarious ones heavily rely on a single compound, dibutyl phthalate (DBP), for sexual communication. DBP is abundantly released by solitary female locusts and elicits strong attraction of male solitary and gregarious locusts. Solitary adult males display much higher electrophysiological responses to DBP than adult females. Additionally, LmigOr13 was identified as the DBP-specific odorant receptor expressed in neurons housed in basiconic sensilla. Male LmigOr13-/- mutants generated by CRISPR/Cas9 have low electrophysiological responses and behavioral attraction to DBP in both laboratory and field cage experiments. Notably, the attractiveness of DBP to male locusts becomes more evident at lower population densities imposed by controlling the cage size. This finding sheds light on the utilization of a sex pheromone to promote reproductive success in extremely low-density conditions and provides important insights into alternative approaches for population monitoring of locusts.

Sexy fingers: Pheromones in the glands of male dendrobatid frogs.

Many animals exchange chemicals during courtship and mating. In some amphibians, sexual chemical communication is mediated by pheromones produced in male breeding glands that are transferred to the female's nostrils during mating. This has been mostly studied in salamanders, despite frogs having similar glands and courtship behaviours suggestive of chemical communication. In Neotropical poison frogs (Dendrobatidae and Aromobatidae), males of many species develop breeding glands in their fingers, causing certain fingers to visibly swell. Many also engage in cephalic amplexus, whereby the male's swollen fingers are placed in close contact with the female's nares during courtship. Here, we investigate the possible roles of swollen fingers in pheromone production using whole-transcriptome sequencing (RNAseq). We examined differential gene expression in the swollen versus non-swollen fingers and toes of two dendrobatid species, Leucostethus brachistriatus and Epipedobates anthonyi, both of which have specialised mucous glands in finger IV, the latter of which has cephalic amplexus. The overwhelming pattern of gene expression in both species was strong upregulation of sodefrin precursor-like factors (SPFs) in swollen fingers, a well-known pheromone system in salamanders. The differentially expressed SPF transcripts in each species were very high (>40), suggesting a high abundance of putative protein pheromones in both species. Overall, the high expression of SPFs in the swollen fingers in both species, combined with cephalic amplexus, supports the hypothesis that these traits, widespread across members of the subfamilies Colostethinae and Hyloxalinae (ca. 141 species), are involved in chemical signalling during courtship.

Muchos animales intercambian sustancias químicas durante el cortejo y el apareamiento. En algunos anfibios, la comunicación química sexual está mediada por feromonas producidas en las glándulas reproductoras de los machos que se transfieren a las hembras durante el apareamiento. Esto se ha estudiado sobre todo en salamandras, a pesar de que las ranas tienen glándulas similares y comportamientos de cortejo que sugieren una comunicación química. En las ranas venenosas neotropicales (Dendrobatidae y Aromobatidae), los machos de muchas especies desarrollan glándulas en los dedos, lo que hace que algunos dedos se vean hinchados. Asimismo, varias especies presentan amplexo cefálico, comportamiento de cortejo en el cual los dedos hinchados entran en estrecho contacto con las narinas y boca de la hembra. En este estudio investigamos las posibles funciones de los dedos hinchados en la producción de feromonas mediante la secuenciación del transcriptoma completo (RNAseq). Examinamos la expresión génica diferencial en los dedos hinchados y no hinchados de dos especies de dendrobátidos, Leucostethus brachistriatus y Epipedobates anthonyi, ambos con glándulas mucosas especializadas en el dedo IV, y esta última especie, con amplexo cefálico. El patrón abrumador de expresión génica en ambas especies fue la alta expression de Sodefrin Precursor­Like Factor (SPF) en los dedos hinchados, un sistema de feromonas ampliamente conocido en las salamandras. El número de transcritos SPF expresados diferencialmente en cada especie fue muy elevado (>40), lo que sugiere una gran abundancia de feromonas proteicas putativas en ambas especies. En general, la elevada expresión de SPF en los dedos hinchados en ambas especies, combinada con el amplexo cefálico, apoya la hipótesis de que estos rasgos, muy extendidos entre los miembros de las subfamilias Colostethinae e Hyloxalinae (aprox 141 especies), están implicados en la señalización química durante el cortejo.

OBP83b and OBP49a Involved in the Perception of Female-Derived Pheromones in Bactrocera dorsalis (Hendel).

In Bactrocera dorsalis, both males and females release chemical signals to attract mates. In our previous study, we identified ethyl laurate, ethyl myristate, and ethyl palmitate as potent female-derived pheromones that contribute to mate attraction. However, the mechanisms underlying the olfactory recognition remain unclear. In this study, we observed strong antennal and behavioral responses in male B. dorsalis to these female-derived pheromones, and further investigation revealed significant upregulation of OBP49a and OBP83b following exposure to these compounds. Through fluorescence competitive binding assays and RNA interference techniques, we demonstrated the crucial roles of OBP49a and OBP83b in detecting female-derived pheromones. Finally, molecular docking analysis identified key residues, including His134 in OBP83b and a lysine residue in OBP49a, which formed hydrogen bonds with female-derived pheromones, facilitating their binding. These findings not only advance our understanding of olfactory recognition of pheromones in B. dorsalis but also offer potential targets for developing olfaction-interfering techniques for pest control.

The novel function of an orphan pheromone receptor reveals the sensory specializations of two potential distinct types of sex pheromones in noctuid moth.

Sex pheromones play crucial role in mating behavior of moths, involving intricate recognition mechanisms. While insect chemical biology has extensively studied type I pheromones, type II pheromones remain largely unexplored. This study focused on Helicoverpa armigera, a representative species of noctuid moth, aiming to reassess its sex pheromone composition. Our research unveiled two previously unidentified candidate type II sex pheromones-3Z,6Z,9Z-21:H and 3Z,6Z,9Z-23:H-in H. armigera. Furthermore, we identified HarmOR11 as an orphan pheromone receptor of 3Z,6Z,9Z-21:H. Through AlphaFold2 structural prediction, molecular docking, and molecular dynamics simulations, we elucidated the structural basis and key residues governing the sensory nuances of both type I and type II pheromone receptors, particularly HarmOR11 and HarmOR13. This study not only reveals the presence and recognition of candidate type II pheromones in a noctuid moth, but also establishes a comprehensive structural framework for PRs, contributing to the understanding of connections between evolutionary adaptations and the emergence of new pheromone types.

Enantioselective Synthesis of the Active Sex Pheromone Components of the Female Lichen Moth, Lyclene dharma dharma, and Their Enantiomers.

The Lichen moth, Lyclene dharma dharma (Arctiidae, Lithosiinae), plays a significant role in forest ecosystem dynamics. A concise and novel method to synthesize the active sex pheromone components, (S)-14-methyloctadecan-2-one ((S)-1), (S)-6-methyloctadecan-2-one ((S)-2), and their enantiomers has been developed. Key steps in the synthesis include the use of Evans' chiral auxiliaries, Grignard cross-coupling reactions, hydroboration-oxidation, and Wacker oxidation. The synthesized sex pheromone components hold potential value for studies on communication mechanisms, species identification, and ecological management.

Interaction between females and males grapevine moth Lobesia botrana modifies further mating preference.

During reproduction, females may boost their fitness by being selective based on direct material benefits provided by the males, such as nuptial gifts. In Lepidoptera, male provides a spermatophore containing nutrients. However, virgin males produce a bigger spermatophore, containing spermatozoa and nutrients, allowing higher female fertility. Lepidoptera females that could detect the sexual status of males may thus prefer a male without previous mating experience (i.e. a virgin male). This mate selection could be achieved by the use of chemical indices, such as sexual pheromones and cuticular compounds, known to be possibly exchanged during reproduction, and which can be indicators of a previous mating experience and known to be possibly sources of information exchanged. In this study, we experimentally presented Lobesia botrana virgin males with females in order for them to be exposed to females' natural sexual pheromones or cuticular compounds. 12 or 48 h after the exposure of males to either females' sexual pheromones or cuticular compounds, these males were confronted to naïve females, which have a choice between them or a virgin non-exposed males. We highlighted that, despite producing a spermatophore of similar volume, all exposed virgin males were less likely to mate with females 12 h after exposure, while after 48 h of exposure this is only the case for virgin males exposed to sexual pheromones. L. botrana females may thus discriminate male sexual experience based on chemical cues (either from cues transferred directly from females to males, or from changes in the cuticular or pheromone males' profile) indicating past mating experiences. Mating duration was longer for males exposed to sexual pheromones after 12 h only, and for males exposed to cuticular compounds after 48 h only. Pheromones signal might be more persistent over time and seems to more easily gather information for males. The physiological reasoning behind this result still needs to be investigated.

Simultaneous mating disruption of two moth pests of the vineyard (Lobesia botrana and Cryptoblabes gnidiella) through a biodegradable sex pheromone dispenser.

Lobesia botrana (Lepidoptera: Tortricidae) and Cryptoblabes gnidiella (Lepidoptera: Pyralidae) represent a threat to wine production in Mediterranean countries. In recent years, the development of new formulations promoted the spread of pheromone-based mating disruption (MD) as an effective tool for the management of several insect pests in different agricultural contexts. In this study, we investigated the efficacy of an experimental dispenser designed for simultaneous MD of these two pests. The biodegradable double-tube dispenser (Isonet® L CG-BIOX235) was tested for two years in two Italian wine-growing sites, the first in Apulia (Southern Italy), and the second in Tuscany (Central Italy). Isonet® L CG-BIOX235 efficacy was evaluated by testing different doses (i.e., 300, 400, and 500 dispensers/ha), on different varieties (i.e., Aglianico, Syrah, and Viognier), and comparing it with an untreated control. The MD performed using this dispenser significantly reduced the infestation of both L. botrana (i.e., percentage of infested bunches and number of nests per bunch) and C. gnidiella compared to the untreated control, although the occurrence of the latter fluctuated throughout the two-year trials. Overall, although our results underline the possibility of combining the pheromones of the two pests in a single dispenser for their simultaneous MD, they also highlight the need for further studies on some aspects of C. gnidiella biology and consequently improve the MD efficacy against this species.

No sexual pheromones in Anopheles mosquitoes?

Swarming behavior is the cornerstone of the anopheline mating system. At dusk, males congregate in monospecific swarms in which females come to find a mate once in their lives. Although many Anopheles species coexist in sympatry, hybrids are infrequent, suggesting the existence of strong premating reproductive barriers. Chemical cues, particularly pheromones, often play a crucial role in bringing sexes together in a species-specific manner among insects. While the existence of sexual pheromones in Anopheles species has been postulated, only a few studies developed experimental designs to investigate their presence. Here, we discuss the contrasting and debatable findings regarding both long-range and contact sex pheromones in the context of swarm ecology in Anopheles species.

Identification of the Sex Pheromone of the Asparagus Moth, Parahypopta Caestrum (Lepidoptera, Cossidae).

Chemical, electrophysiological, and field trapping experiments were carried out to identify the female-produced sex pheromone of the asparagus moth, Parahypopta caestrum, a very serious pests of asparagus cultivations in southern Europe. Gas chromatography coupled with mass spectrometry and electroantennogram detection (GC-MS-EAD) analysis of hexane and solid-phase microextraction (SPME) extracts of sex pheromone glands of calling females consistently detected four compounds eliciting EAG responses in male moth antennae. According to their GC retention times, mass spectra, and comparative EAG analyses with reference standards, these EAD-active compounds were identified as (Z)-9-tetradecenol (Z9-14:OH), (Z)-5-tetradecenyl acetate (Z5-14:Ac), (Z)-7-tetradecenyl acetate (Z7-14:Ac), and (Z)-9-tetradecenyl acetate (Z9-14:Ac), respectively. In the SPME extracts from the head-space of individual abdominal tips, Z9-14:Ac, Z5-14:Ac, Z7-14:Ac, and Z9:14 OH were detected in the ratio of 82:9:5:4. In EAG dose-response experiments, Z9-14:Ac was the strongest antennal stimulant at different doses tested. In field trapping experiments, Z9-14:Ac, Z7-14:Ac, and Z5-14:Ac proven to be essential for male attraction and a their 85:5:10 blend loaded onto green rubber septum dispensers was significantly more effective than single-, two-, and any other three-component blend of these compounds. The addition of Z9-14:OH to the optimal blend resulted in a significant reduction of male catches. The attractive blend here identified allowed for an effective and accurate monitoring of P. caestrum flight activity in southern Italy.