Protocol for aerial trapping and analyses of candidate pheromone compounds released by moths via gas chromatography-mass spectrometry.

Detection of pheromones is pivotal to chemical ecology and agronomy; however, analytic detection of the volatile pheromone components from odorized air is highly challenging. Here, we introduce a protocol for the detection of airborne pheromones from female moths, which are key models for chemosensory studies. We describe a step-by-step guide from pheromone collection to quantitative estimation of pheromone components. We also detail procedures for gas chromatography-mass spectrometry (GC-MS) analysis. This protocol has potential applications beyond chemosensory research, particularly in environmental chemistry. For complete details on the use and execution of this protocol, please refer to Ghosh et al.1.

Two Structural Analogs of Kairomones are Detected by an Odorant Receptor HvarOR28 in the Coccinellid Hippodamia variegata.

In natural environments, general plant volatiles and herbivore-induced plant volatiles (HIPVs) serve as critical clues for predatory natural enemies in the search for prey. The insect olfactory system plays a vital role in perceiving plant volatiles including HIPVs. In this study, we found that HIPV (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) and the plant volatile geranyl acetate (GA), two structurally similar chemicals, displayed electrophysiological activities on the antennae of the ladybird Hippodamia variegata, but were only attractive to adult females in behavior. Moreover, mated female ladybirds laid a significantly higher number of eggs on TMTT-treated and GA-treated cotton leaves compared to controls. Screening of female-biased odorant receptors (ORs) from the antennal transcriptomes, performing Xenopus oocytes expression coupled with two-electrode voltage clamp recordings, suggested that HvarOR28 specifically tuned to TMTT and GA. Molecular docking and site-directed mutagenesis revealed that the amino acid residues Tyr143 and Phe81 of HvarOR28 are the key site for binding with TMTT and GA. Furthermore, RNA interference (RNAi) assay demonstrated that HvarOR28-silenced individuals demonstrated a notable decrease in electrophysiological responses, even female adults almost lost behavioral preference for the two compounds. Thus, it could be concluded that HvarOR28 in H. variegata contributes to facilitating egg laying through the perception of TMTT and GA. These findings may help to develop new olfactory modulators based on the behaviorally active ligands of HvarOR28.

Design of Novel Membranes for the Efficient Separation of Bee Alarm Pheromones in Portable Membrane Inlet Mass Spectrometric Systems.

Bee alarm pheromones are essential molecules that are present in beehives when some threats occur in the bee population. In this work, we have applied multilevel modeling techniques to understand molecular interactions between representative bee alarm pheromones and polymers such as polymethyl siloxane (PDMS), polyethylene glycol (PEG), and their blend. This study aimed to check how these interactions can be manipulated to enable efficient separation of bee alarm pheromones in portable membrane inlet mass spectrometric (MIMS) systems using new membranes. The study involved the application of powerful computational atomistic methods based on a combination of modern semiempirical (GFN2-xTB), first principles (DFT), and force-field calculations. As a fundamental work material for the separation of molecules, we considered the PDMS polymer, a well-known sorbent material known to be applicable for light polar molecules. To improve its applicability as a sorbent material for heavier polar molecules, we considered two main factors-temperature and the addition of PEG polymer. Additional insights into molecular interactions were obtained by studying intrinsic reactive properties and noncovalent interactions between bee alarm pheromones and PDMS and PEG polymer chains.

Practical preparation of unsaturated very-long-chain fatty acids (VLCFAs) and very-long-chain alkene pollinator attractants.

To prepare very-long-chain fatty acids and alkenes (VLCFAs and VLC alkenes) that are known pollinator attractants for sexually deceptive orchids, and biosynthetic precursors thereof, we applied a methodology allowing us to prepare monounsaturated VLCFAs with chain lengths up to 28 carbons and VLC alkenes up to 31 carbons. We implemented a coupling reaction between commercially available terminal alkynes and bromoalkanoic acids to prepare VLCFAs, allowing the products to be formed in two steps. For VLC alkenes, with many alkyltriphenylphosphonium bromides commercially available, we applied a Wittig reaction approach to prepare (Z)-configured monoenes in a single step. Using practical methods not requiring special reagents or equipment, we obtained 11 VLCFAs in > 90% isomeric purity, and 17 VLC alkenes in > 97% isomeric purity. Such general and accessible synthetic methods are essential for chemical ecology and biochemistry research to aid researchers in unambiguously identifying isolated semiochemicals and their precursors.

Identification and Functional Analysis of Odorant Binding Proteins in Apriona germari (Hope).

Apriona germari (Hope) presents a significant threat as a dangerous wood-boring pest, inflicting substantial harm to forest trees. Investigating the olfactory sensory system of A. germari holds substantial theoretical promise for developing eco-friendly control strategies. To date, however, the olfactory perception mechanism in A. germari remains largely unknown. Therefore, we performed transcriptome sequencing of A. germari across four distinct body parts: antennae, foreleg tarsal segments, mouthparts (maxillary and labial palps), and abdomen terminals, pinpointing the odorant binding protein (OBP) genes and analyzing their expression. We found eight AgerOBPs (5, 19, 23, 25, 29, 59, 63, 70) highly expressed in the antennae. In our competitive binding experiments, AgerOBP23 showed strong binding abilities to the pheromone component fuscumol acetate, eight plant volatiles (farnesol, cis-3-hexenal, nerolidol, myristol acetate, cis-3-hexenyl benzoate, (-)-α-cedrene, 3-ethylacetophenone, and decane), and four insecticides (chlorpyrifos, phoxim, indoxacarb, and cypermethrin). However, AgerOBP29 and AgerOBP63 did not show prominent binding activities to these tested chemicals. Through homology modeling and molecular docking, we identified the key amino acid sites involved in the binding process of AgerOBP23 to these ligands, which shed light on the molecular interactions underlying its binding specificity. Our study suggests that AgerOBP23 may serve as a potential target for future investigations of AgerOBP ligand binding. This approach is consistent with the reverse chemical ecology principle, establishing the groundwork for future studies focusing on attractant or repellent development by exploring further the molecular interactions between OBP and various compounds.

Insects' perception and behavioral responses to plant semiochemicals.

Insect-plant interactions are shaped by the exchange of chemical cues called semiochemicals, which play a vital role in communication between organisms. Plants release a variety of volatile organic compounds in response to environmental cues, such as herbivore attacks. These compounds play a crucial role in mediating the interactions between plants and insects. This review provides an in-depth analysis of plant semiochemicals, encompassing their classification, current understanding of extraction, identification, and characterization using various analytical techniques, including gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) spectroscopy, and infrared (IR) spectroscopy. The article also delves into the manner in which insects perceive and respond to plant semiochemicals, as well as the impact of environmental factors on plant odor emission and insect orientation. Furthermore, it explores the underlying mechanisms by which insects perceive and interpret these chemical cues, and how this impacts their behavioral responses, including feeding habits, oviposition patterns, and mating behaviors. Additionally, the potential applications of plant semiochemicals in integrated pest management strategies are explored. This review provides insight into the intricate relationships between plants and insects mediated by semiochemicals, highlighting the significance of continued research in this field to better understand and leverage these interactions for effective pest control.

Synthetic Alfalfa Infusion Odour Attracts Gravid Culex quinquefasciatus Under Laboratory Conditions.

Gravid culicine mosquitoes rely on olfactory cues for selecting breeding sites containing organic detritus. While this capacity of the mosquitoes is used for surveillance and control, the current methodology is unwieldy, unreliable and expensive in time and labour. This study evaluated the dose-dependent attraction and oviposition response of gravid Culex quinquefasciatus to alfalfa infusions. Through combined chemical and electrophysiological analyses, bioactive volatile organic compounds (VOCs) in the headspace of alfalfa infusions, eliciting attraction, were identified. While phenolic and indolic compounds were the most abundant bioactive VOCs, additional VOCs, including a monoterpene, were required to elicit a significant behavioural response to the synthetic odour blend of alfalfa infusions. Comparative analysis with the commercially available mosquito oviposition pheromone (MOP) was also conducted demonstrating that this standardised synthetic alfalfa infusion odour blend offers a promising lure for targeted surveillance and control of Culex mosquitoes, which may contribute to disease prevention and public health protection.

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.

Geosmin, a Food- and Water-Deteriorating Sesquiterpenoid and Ambivalent Semiochemical, Activates Evolutionary Conserved Receptor OR11A1.

Geosmin, a ubiquitous volatile sesquiterpenoid of microbiological origin, is causative for deteriorating the quality of many foods, beverages, and drinking water, by eliciting an undesirable "earthy/musty" off-flavor. Moreover, and across species from worm to human, geosmin is a volatile, chemosensory trigger of both avoidance and attraction behaviors, suggesting its role as semiochemical. Volatiles typically are detected by chemosensory receptors of the nose, which have evolved to best detect ecologically relevant food-related odorants and semiochemicals. An insect receptor for geosmin was recently identified in flies. A human geosmin-selective receptor, however, has been elusive. Here, we report on the identification and characterization of a human odorant receptor for geosmin, with its function being conserved in orthologs across six mammalian species. Notably, the receptor from the desert-dwelling kangaroo rat showed a more than 100-fold higher sensitivity compared to its human ortholog and detected geosmin at low nmol/L concentrations in extracts from geosmin-producing actinomycetes.

Identification of Semiochemical Candidates Involved in Glossina Palpalis Gambiensis Larviposition Site Selection and Behavioural Responses of Adult Gravid Females.

Tsetse flies (Diptera: Glossinidae) are the cyclical vectors of human and animal trypanosomes. This viviparous insect develops and produces a single larva at 10-day intervals deposited in specific sites. In some species aggregation of larvae has been shown and seems to be mediated by both physical factors and volatile semiochemicals of larval origin. In this context, this study aims to identify chemicals emitted during the pupariation process in Glossina palpalis gambiensis. Volatile Organic Compounds (VOCs) emitted by larvae were identified using static headspace solid-phase microextraction and gas-chromatography mass-spectrometry (GC-MS) analysis. Electrophysiology and behavioural assays were performed on gravid females to confirm VOCs behavioural activity and attractiveness. GC-MS results revealed ten chemicals emitted during the pupariation process of G. p. gambiensis larvae. Among these chemicals, gravid females were shown to detect nine of them during coupled gas chromatography - electroantennographic detection tests. Behavioural assays highlighted two compounds were as attractive as pupae and one compound and a blend of four compounds were more attractive than pupae. Although the larval origin of some of them needs to be confirmed as they may also likely produced by micro-organisms, these compounds induced significant behavioural responses in the laboratory. Further experiments have to explore the biological activity and competitiveness of these compounds in the field. This work opens interesting opportunities for behavioural manipulation and control of tsetse flies.

Pre-treatment of Cucurbita maxima 'Hokkaido orange' by Viscum album aqueous extracts in search of allelopathic potential.

Viscum album L. (VA) is a unique plant with regard to its biological content. It is rich in many different metabolites with high potential in various spheres of human activity. We conducted a pilot study with 5 VA aqueous extracts of different host-tree species for pre-sowing treatment of Cucurbita maxima 'Hokkaido orange' seeds. We set the following objectives consisting of hypotheses (1) H01 is based on different effects of tested VA extracts depending on host trees and time of pre-treatment; (2) H02 focuses on the allopathic properties of the tested extracts affecting the plant growth and development by dose-response relationship; (3) A01 considers highly biologically active compounds of VA extracts also containing allelochemicals that can be used to regulate plant growth processes and create eco-friendly and resilient cities. The analysis of the stimulatory allelopathy index for 7 parameters demonstrates the direct effect of VA extracts in 62.3% of cases. The variability of the broad spectrum of effects of VA extracts of different host trees on the ontogenesis of C. maxima plants shows the presence of potential allelochemicals, resulting from the vital products of the host-parasite relationship. These effects are not fully explained by total polyphenol content and antioxidant activity as in previous studies of other mistletoe species. The authors consider this work a pilot study that expands the areas of application of VA extracts and knowledge about potential sources of allelochemicals.

Chemical defence of a centipede (Clinopodes flavidus).

Chemical substances are of utmost importance for the biotic interactions between animals and their predators/parasites; many of these semiochemicals are emitted for defence purposes. One of the most deterrent and toxic biogenic substances we know of is hydrogen cyanide, which can be stored by certain insects, millipedes, centipedes and arachnids in the form of stable and less volatile molecules. The aim of this study was to analyse the biology and chemistry of such a defence mechanism in a geophilomorph centipede (Chilopoda). The cyanogenic secretion of Clinopodes flavidus is discharged from the ventral glands, whose glandular units are located in the space between the cuticle and the trunk muscles and do not extend deep into the segment. In addition to hydrogen cyanide, the ventral secretion contains 2-methylpentanoic acid, benzaldehyde, benzoyl cyanide, 2-methyl branched C-9 carboxylic acid (tentatively identified as 2-methyloctanoic acid), methyl 2-phenylacetate, benzoic acid and mandelonitrile as well as four major proteins with a molecular weight of 150, 66.2, 59 and 55 kDa. The correlation between the presence of ventral glands and guarding with the female's ventral side facing away from the eggs and young indicates a functional link between these two traits. We hope that the specificity of the chemical composition of the ventral secretion could serve as a criterion for chemotaxonomy and that the analysis of more species will help to clarify the phylogenetic relationships within the Geophilomorpha.

Protein-Ligand Binding and Structural Modelling Studies of Pheromone-Binding Protein-like Sol g 2.1 from Solenopsis geminata Fire Ant Venom.

Sol g 2 is the major protein in Solenopsis geminata fire ant venom. It shares the highest sequence identity with Sol i 2 (S. invicta) and shares high structural homology with LmaPBP (pheromone-binding protein (PBP) from the cockroach Leucophaea maderae). We examined the specific Sol g 2 protein ligands from fire ant venom. The results revealed that the protein naturally formed complexes with hydrocarbons, including decane, undecane, dodecane, and tridecane, in aqueous venom solutions. Decane showed the highest affinity binding (Kd) with the recombinant Sol g 2.1 protein (rSol g 2.1). Surprisingly, the mixture of alkanes exhibited a higher binding affinity with the rSol g 2.1 protein compared to a single one, which is related to molecular docking simulations, revealing allosteric binding sites in the Sol g 2.1 protein model. In the trail-following bioassay, we observed that a mixture of the protein sol g 2.1 and hydrocarbons elicited S. geminata worker ants to follow trails for a longer time and distance compared to a mixture containing only hydrocarbons. This suggests that Sol g 2.1 protein may delay the evaporation of the hydrocarbons. Interestingly, the piperidine alkaloids extracted have the highest attraction to the ants. Therefore, the mixture of hydrocarbons and piperidines had a synergistic effect on the trail-following of ants when both were added to the protein.

A Hitchhiker's Ride: The Honey Bee Louse Braula Coeca (Diptera: Braulidae) Selects its Host by Eavesdropping.

The bee louse Braula spp. had until recently a distribution coincident with its host the honey bee. The adult fly usually attaches to a worker honey bee and steals food from its mouth. However, not all worker bees carry Braula spp. and the mechanism used by Braula spp. to select hosts is not well understood. Using choice remounting bioassays and chemical analyses, we determined host selection and the cues used by B. coeca, a species associated with the African honey bee Apis mellifera scutellata. Braula coeca successfully remounted bees from which they were initially removed and preferred their mandibular gland pheromones (MDG) over those of bees not carrying them. The bee lice did not show any preference for the cuticular hydrocarbons of both types of workers. Chemical analyses of the MDG extracts, revealed quantitative differences between the two categories of workers, with workers carrying B. coeca having more of the queen substance (9-oxo-2(E)-decenoic acid) and worker substance (10-hydroxy-2(E)-decenoic). Braula coeca showed a dose response to the queen substance, indicating its ability to use host derived kairomones as cues that allowed it to benefit from trophallactic dominance by individuals that have a higher probability of being fed by other workers.

(R)-(+)-γ-Decalactone is Conserved in North America as a Pheromone Component of Osmoderma eremicola (Coleoptera: Scarabaeidae) and a Kairomone of Elater abruptus (Coleoptera: Elateridae).

The scarab genus Osmoderma (Coleoptera: Scarabaeidae) includes several large species called hermit beetles that develop within dead and decaying hardwood trees. Males of at least three Palearctic species produce the aggregation-sex pheromone (R)-(+)-γ-decalactone, including the endangered O. eremita (Scopoli). However, hermit beetles have received less attention in the western hemisphere, resulting in a large gap in our knowledge of the chemical ecology of Nearctic species. Here, we identify (R)-( +)-γ-decalactone as the primary component of the aggregation-sex pheromone of the North American species Osmoderma eremicola (Knoch). Field trials at sites in Wisconsin and Illinois revealed that both sexes were attracted to lures containing (R)-(+)-γ-decalactone or the racemate, but only males of O. eremicola produced the pheromone in laboratory bioassays, alongside an occasional trace of the chain-length analog γ-dodecalactone. Females of the congener O. scabra (Palisot de Beauvois) were also significantly attracted by γ-decalactone, suggesting further conservation of the pheromone, as were females of the click beetle Elater abruptus Say (Coleoptera: Elateridae), suggesting that this compound may have widespread kairomonal activity. Further research is needed to explore the behavioral roles of both lactones in mediating behavioral and ecological interactions among these beetle species.

Allelochemicals from the seaweeds and their bioprospecting potential.

Allelochemicals are secondary metabolites which function as a natural protection against grazing activities by algae and higher plants. They are one of the major metabolites engaged in the interactions of organisms. The chemically mediated interactions between organisms significantly influence the functioning of the ecosystems. Most of these compounds are secondary metabolites comprising sterols, terpenes, and polyphenols. These compounds not only play a defensive role, but also exhibit biological activities such as antioxidants, anti-cancer, anti-diabetes, anti-inflammation, and anti-microbial properties. This review article discusses the current understanding of the allelochemicals of seaweeds and their bioprospecting potential that can bring benefit to humanity. Specifically, the bioactive substances having specific health benefits associated with the consumption or application of seaweed-derived compounds. The properties of such allelochemicals can have implications for bioprospecting pharmaceutical, nutraceutical and cosmetic applications.

Structure, Synthesis, and Bioassays of Sex Pheromone for Pyrausta machaeralis (Lepidoptera: Crambidae).

The leaf skeletonizer, Pyrausta machaeralis (Lepidoptera: Crambidae), is a serious insect pest of teak (Tectona grandis) in China. The application of insect pheromones is widely applied as an environmentally friendly technology for integrated pest management (IPM). In the present study, crude extracts of sex pheromone glands of calling P. machaeralis females were collected and then analyzed using gas chromatography/electroantennographic detection (GC/EAD) and gas chromatography-mass spectrometry (GC-MS). The combination of infrared spectroscopy (IR) and nuclear magnetic resonance (NMR) spectrometry was used for structure identification. Afterward, their electrophysiological and behavioral activity was evaluated in the laboratory and field. Herein, we eventually determined two active components, E-11-tetradecenyl acetate (E11-14:Ac) and Z-11-tetradecenyl acetate (Z11-14:Ac), at a ratio of 96:4, as the sex pheromone of P. machaeralis. The identification of sex pheromones would facilitate the development of efficient strategies for monitoring and controlling the field populations of P. machaeralis.

An acyl-CoA thioesterase is essential for the biosynthesis of a key dauer pheromone in C. elegans.

Methyl ketone (MK)-ascarosides represent essential components of several pheromones in Caenorhabditis elegans, including the dauer pheromone, which triggers the stress-resistant dauer larval stage, and the male-attracting sex pheromone. Here, we identify an acyl-CoA thioesterase, ACOT-15, that is required for the biosynthesis of MK-ascarosides. We propose a model in which ACOT-15 hydrolyzes the ß-keto acyl-CoA side chain of an ascaroside intermediate during ß-oxidation, leading to decarboxylation and formation of the MK. Using comparative metabolomics, we identify additional ACOT-15-dependent metabolites, including an unusual piperidyl-modified ascaroside, reminiscent of the alkaloid pelletierine. The ß-keto acid generated by ACOT-15 likely couples to 1-piperideine to produce the piperidyl ascaroside, which is much less dauer-inducing than the dauer pheromone, asc-C6-MK (ascr#2, 1). The bacterial food provided influences production of the piperidyl ascaroside by the worm. Our work shows how the biosynthesis of MK- and piperidyl ascarosides intersect and how bacterial food may impact chemical signaling in the worm.

Cuticular lipid profiles of selected species of cyclocephaline beetles (Melolonthidae, Cyclocephalini).

Neotropical cyclocephaline beetles, a diverse group of flower-loving insects, significantly impact natural and agricultural ecosystems. In particular, the genus Cyclocephala, with over 350 species, displays polymorphism and cryptic complexes. Lacking a comprehensive DNA barcoding framework, accessible tools for species differentiation are needed for research in taxonomy, ecology, and crop management. Moreover, cuticular hydrocarbons are believed to be involved in sexual recognition mechanisms in these beetles. In the present study we examined the cuticular chemical profiles of six species from the genus Cyclocephala and two populations of Erioscelis emarginata and assessed their efficiency in population, species, and sex differentiation. Overall we identified 74 compounds in cuticular extracts of the selected taxa. Linear alkanes and unsaturated hydrocarbons were prominent, with ten compounds between them explaining 85.6% of species dissimilarity. Although the cuticular chemical profiles efficiently differentiated all investigated taxa, only C. ohausiana showed significant cuticular profile differences between sexes. Our analysis also revealed two E. emarginata clades within a larger group of 'Cyclocephala' species, but they were not aligned with the two studied populations. Our research underscores the significance of cuticular lipid profiles in distinguishing selected cyclocephaline beetle species and contemplates their potential impact as contact pheromones on sexual segregation and speciation.