Pheromone-binding Proteins Research Articles

Comparation of pheromone-binding proteins 1 and 2 of Spodoptera frugiperda in perceiving the three sex pheromone components Z9-14:Ac, Z7-12: Ac and Z11-16: Ac

Pheromone-binding proteins (PBPs) are mainly responsible for binding and transporting hydrophobic pheromone molecules across the aqueous sensilla lymph to the receptor proteins. The preference of each PBP is believed to be different for each pheromone component within a single species. Significantly higher expression level of PBP1 and PBP2 in the male antennae of Spodoptera frugiperda suggesting that SfruPBP1 and SfruPBP2 might play important roles in pheromone perception. However, the preference of these two PBP to the three main pheromone components Z9-14: Ac, Z7-12: Ac and Z11-16: Ac have not been determined. In this study, a fluorescence competitive binding assay revealed that the binding intensities of SfruPBP1 and SfruPBP2 to Z9-14: Ac or Z7-12: Ac was comparable. We then used the CRISPR/Cas9 system to individually or simultaneously knock out PBP1 and PBP2 in S.frugiperda. The result of courtship behavior indicated that SfruPBP1 and SfruPBP2 were indispensable and played equal roles in perceiving the pheromones Z9-14: Ac and Z7-12: Ac for orientation, wing vibration, and hair-pencil display. Compared with Z9-14:Ac and Z7-12: Ac, Z11-16: Ac showed higher or medium binding intensities with SfruPBP1 and SfruPBP2 but played a minor role in inducing the wing vibration behavior. The results of this study are valuable for elucidating the mechanisms involved in sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques.

Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in Mythimna loreyi (Lepidoptera: Noctuidae).

Quantitative real-time PCR (qRT-PCR) is a widely applied technique for accurately assessing the expression of target genes. In practice, the evaluation of gene expression requires appropriate reference genes. To screen reliable reference genes for evaluating gene expression via qRT-PCR in Mythimna loreyi, a notorious migratory pest across Asia, Africa, Europe, and Australia, we assessed the expression stability of 13 candidate reference genes in M. loreyi using the ΔCt method, BestKeeper, Normfinder, GeNorm, and the web-based comprehensive platform RefFinder. These reference genes include RPL10, RPL27, RPL32, RPS3, TATA-box, GAPDH, AK, Actin, EF, α-tubulin, SOD, 18S rRNA, and FTZ-F1, which is frequently employed in Lepidoptera insects. Our findings revealed that the performance of the candidate reference gene depended on experimental conditions. Specifically, RPL27 and RPL10 were the most suitable for evaluating expression changes across developmental stages, tissues, and adult ages. The optimal reference genes were recommended in specific experiment conditions, for instance, EF and RPS3 were recommended for mating status, AK and RPL10 were recommended for temperature treatments, RPL27 and FTZ-F1 were recommended for larva diet, and EF and RPL27 were recommended for adult diet treatments. Additionally, expression profiles of pheromone-binding protein 2 (MlorPBP2) and glutathione S-transferase (MlorGST1) were used to validate the reference genes. This study provides reference genes for the accurate normalization of qRT-PCR data, laying the groundwork for studying the expression of target genes in M. loreyi.

EsigPBP3 Was the Important Pheromone-Binding Protein to Recognize Male Pheromones and Key Eucalyptus Volatiles.

Pheromone-binding proteins (PBPs) are specific odorant-binding proteins that can specifically recognize insect pheromones. Through transcriptional analysis of the antennae of adult Endoclita signifer, EsigPBP3 was discovered and identified, and EsigPBP3 was found to be highly expressed in the antennae of male moths. Based on the binding characteristics and ability of EsigPBP3, we can find the key ligands and binding site to consider as a target to control the key wood bore E. signifier. In this study, the fluorescence competitive binding assays (FCBA) showed that EsigPBP3 had a high binding affinity for seven key eucalyptus volatiles. Molecular docking analysis revealed that EsigPBP3 had the strongest binding affinity for the sexual pheromone component, (3E,7E)-4,7,11-trimethyl-1,3,7,10-dodecatetraene. Furthermore, same as the result of FCBA, the EsigPBP3 exhibited high binding affinities to key eucalyptus volatiles, eucalyptol, α-terpinene, (E)-beta-ocimene, (-)-β-pinene, and (-)-α-pinene, and PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 are key sites. In summary, EsigPBP3 exhibits high binding affinity to male pheromones and key volatile compounds and the crucial binding sites PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 can act as targets in the recognition of E. signifier pheromones.

Binding characteristics of pheromone-binding protein 1 in Glyphodes pyloalis to organophosphorus insecticides: Insights from computational and experimental approaches.

Glyphodes pyloalis (Lepidoptera: Pyralidae) is one of the major pests in mulberry production in China, which has developed resistance to various insecticides. Chemoreception is one of the most crucial physiological tactics in insects, playing a pivotal role in recognizing chemical stimuli in the environment, including noxious stimuli such as insecticides. Herein, we obtained recombinant pheromone-binding protein 1 (GpylPBP1) that exhibited antennae-biased expression in G. pyloalis. Ligand-binding assays indicated that GpylPBP1 had the binding affinities to two organophosphorus insecticides, with a higher binding affinity to chlorpyrifos than to phoxim. Computational simulations showed that a mass of nonpolar amino acid residues formed the binding pocket of GpylPBP1 and contributed to the hydrophobic interactions in the bindings of GpylPBP1 to both insecticides. Furthermore, the binding affinities of three GpylPBP1 mutants (F12A, I52A, and F118A) to both insecticides were all significantly reduced compared to those of the GpylPBP1-wild type, suggesting that Phe12, Ile52, and Phe118 residues were crucial binding sites and played crucial roles in the bindings of GpylPBP1 to both insecticides. Our findings can be instrumental in elucidating the effects of insecticides on olfactory recognition in moths and facilitating the development of novel pest management strategies using PBPs as targets based on insect olfaction.

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.

Molecular and Functional Characterization of Pheromone Binding Protein 2 from Cyrtotrachelus buqueti (Coleoptera: Curculionidae)

Pheromone-binding proteins (PBPs) play important roles in binding and transporting sex pheromones. However, the PBP genes identified in coleopteran insects and their information sensing mechanism are largely unknown. Cyrtotrachelus buqueti (Coleoptera: Curculionidae) is a major insect pest of bamboo plantations. In this study, a novel PBP gene, CbuqPBP2, from C. buqueti was functionally characterized. CbuqPBP2 was more abundantly expressed in the antennae of both sexes than other body parts, and its expression level was significantly male-biased. Fluorescence competitive binding assays showed that CbuqPBP2 exhibited the strongest binding affinity to dibutyl phthalate (Ki = 6.32 μM), followed by styrene (Ki = 11.37 μM), among twelve C. buqueti volatiles. CbuqPBP2, on the other hand, showed high binding affinity to linalool (Ki = 10.55), the main volatile of host plant Neosinocalamus affinis. Furthermore, molecular docking also demonstrated the strong binding ability of CbuqPBP2 to dibutyl phthalate, styrene, and linalool, with binding energy values of −5.7, −6.6, and −6.0 kcal/mol, respectively, and hydrophobic interactions were the prevailing forces. The knockdown of CbuqPBP2 expression via RNA interference significantly reduced the electroantennography (EAG) responses of male adults to dibutyl phthalate and styrene. In conclusion, these results will be conducive to understanding the olfactory mechanisms of C. buqueti and promoting the development of novel strategies for controlling this insect pest.

A Study of Adult Olfactory Proteins of Primitive Ghost Moth, Endoclita signifer (Lepidoptera, Hepialidae).

Endoclita signifer is a prominent wood-boring insect species in eucalyptus plantations in Guangxi, China, causing significant ecological and economic damage. A novel approach to controlling the challenging wood-boring pest involves disrupting the olfactory communication between insects and the volatile compounds emitted by plants. To identify the olfactory proteins contributing to host selection based on 11 GC-EAD-active volatiles from eucalyptus leaves and to discover the highly expressed olfactory proteins, we conducted a study on the antennal transcriptomes of adult E. signifer and screened key olfactory proteins in the antennae. We identified a total of 69 olfactory proteins. When compared to the larval transcriptomes, the antennal transcriptome of adult E. signifer revealed the presence of 17 new odorant-binding proteins (OBPs), including 2 pheromone-binding proteins (PBPs), 7 previously unreported chemosensory proteins (CSPs), 17 new odorant receptors (ORs), 4 new gustatory receptors (GRs), 11 novel ionotropic receptors (IRs), and 2 sensory neuron membrane proteins (SNMPs). Through the phylogenetic tree of OBPs and ORs, we identified EsigPBP2 and EsigPBP3 as two of the three PBPs, designated EsigOR13 as EsigOrco, and recognized EsigOR10 and EsigOR22 as the newly discovered EsigPRs in E. signifer. In the adult antennae, the expression levels of EsigGOBP14, EsigGOBP13, EsigOBP14, EsigOBP17, EsigCSP14, and EsigOR16 were notably high, indicating that these proteins could be pivotal in binding to plant volatiles.

High-efficiency removal of pyrethroids using a redesigned odorant binding protein

Pyrethroids are ubiquitously present in environmental media and threaten both the ecosystem and human health. To explore effective ways to remove pyrethroids from the environment, an odorant binding protein (OBP) with affinity for various pyrethroids was investigated. Initially, the target OBP, Spodoptera littoralis pheromone binding protein 1 (SlitPBP1), underwent redesign to enhance its affinity for pyrethroids. The modified SlitPBP1E97ND106E demonstrated a substantially increased affinity for deltamethrin (DeltaM), with a dissociation constant of 0.77 ± 0.17 μM. The affinity of SlitPBP1E97ND106E for other pyrethroids also increased to varying extents. Consequently, SlitPBP1E97ND106E displayed a markedly enhanced capability to adsorb and remove pyrethroids. When exposed to free SlitPBP1E97ND106E in solution, the reduction in DeltaM surged from 16.78 ± 0.32% to 97.51 ± 0.56%. SlitPBP1E97ND106E was immobilized by coupling the protein to Ni2+-NTA agarose resin. Liquid chromatography results attested to the superior efficacy of immobilized SlitPBP1E97ND106E in removing pyrethroids, especially DeltaM. No significant differences in pyrethroid removal were detected across various water samples. Our findings introduce a potent tool for pyrethroid removal. A wider range of OBPs can similarly be optimized and applied to remove organic pollutants, including but not limited to pesticides.

Molecular mechanism of sex pheromone perception in male Mythimna loreyi revealed by in vitro system.

Mythimna loreyi is an important agricultural pest with a sensitive sex pheromone communication system. To clarify the pheromone binding proteins (PBPs) and pheromone receptors (PRs) involved in sex pheromone perception is important for both understanding the molecular olfactory mechanism and developing a new pest control strategy in M. loreyi. First, the electroantennogram (EAG) assay showed that male M. loreyi displayed the highest response to the major sex pheromone component Z9-14:Ac, and higher responses to two minor components, Z7-12:Ac and Z11-16:Ac. Second, the fluorescence competition binding assay showed that PBP1 bound all three pheromones and other tested compounds with high or moderate affinity, while PBP2 and PBP3 each bound only one pheromone component and few other compounds. Third, functional study using the Xenopus oocyte system demonstrated that, of the six candidate PRs, PR2 was weakly sensitive to the major pheromone Z9-14:Ac, but was strongly sensitive to pheromone analog Z9-14:OH; PR3 was strongly and specifically sensitive to a minor component Z7-12:Ac; PR4 and OR33 were both weakly sensitive to another minor component, Z11-16:Ac. Finally, phylogenetic relationship and ligand profiles of PRs were compared among six species from two closely related genera Mythimna and Spodoptera, suggesting functional shifts of M. loreyi PRs toward Spodoptera PRs. Functional differentiations were revealed among three PBPs and six PRs in sex pheromone perception, laying an important basis for understanding the molecular mechanism of sex pheromone perception and for developing new control strategies in M. loreyi. © 2023 Society of Chemical Industry.

Antennal transcriptome analysis of odorant-binding proteins and characterization of GOBP2 in the variegated cutworm Peridroma saucia.

Odorant-binding proteins (OBPs) are expressed at extremely high concentrations in the chemo-sensilla lymph of insects and have long been thought to be crucial for delivering the semiochemicals to the odorant receptors. They are represented by multiple classes: general odorant-binding proteins (GOBP1 and GOBP2) and pheromone-binding proteins. In the current study, we identified a total of 35 OBPs in the antennal transcriptome of Peridroma saucia, a worldwide pest that causes serious damage to various crops. A gene expression value (TPM, transcripts per million) analysis revealed that seven OBPs (PsauPBP1/2/3, PsauGOBP1/2, PsauOBP6, and PsauOBP8) were highly abundant in the antennae. Next, we focused on the expression and functional characterization of PsauGOBP2. Real-time quantitative-PCR analysis demonstrated that PsauGOBP2 was predominantly expressed in the antennae of both sexes. Fluorescence binding assays showed that the recombinant PsauGOBP2 strongly binds to the female sex pheromone components Z11-16: Ac (Ki = 4.2μM) and Z9-14: Ac (Ki = 4.9μM) and binds moderately (6µM ≤ Ki ≤ 13µM) to the host plant volatiles phenylethyl acetate, β-myrcene, and dodecanol. Further 3D structural modeling and molecular docking revealed that several crucial amino acid residues are involved in ligand binding. The results not only increase our understanding of the olfactory system of P. saucia but also provide insights into the function of PsauGOBP2 that has implications for developing sustainable approaches for P. saucia management.

Red palm weevil olfactory proteins annotated from the rostrum provide insights into the essential role in chemosensation and chemoreception

Red palm weevil (RPW), Rhynchophorus ferrugineus (Coleoptera: Curculionidae), is rapidly infesting palm trees (Arecaceae) in several countries, threatening coconut, date, and oil cultivations. The male-produced aggregation pheromone in palm weevils has been reported to be secreted through the mouth to the rostrum, a snout-like projection key for pheromone emission and dispersion. The olfactory mechanisms that underlie peripheral odorant detection in RPW have been addressed at the antennal level. However, the rostrum remains unexplored. Through RNA-seq, 27 odorant-binding proteins (OBPs), 6 chemosensory proteins (CSPs), 4 sensory neuron membrane proteins (SNMPs), 21 gustatory receptors (GRs), 25 odorant receptors (ORs) (including one odorant receptor coreceptor, Orco) and 10 ionotropic receptors (IRs), were identified. We reported 27 novel rostrum-specific olfactory proteins (4 IRs, 11 GRs, 2 CSPs, 3 OBPs, and 7 ORs) in R. ferrugineus (Rfer). The OBPs (RferSOBPs) [Rfer with “S” indicating “snout” (rostrum)] were the most abundant transcripts compared with the rest of the olfactory proteins. We identified several rostrum OBPs, which predominately emerged through gene duplication, and were found expressed in both rostrum and antennae. Noticeably, we found R. ferrugineus pheromone-binding protein (RferOBP1768) paralog in the rostrum (RferOBP14) and mapped it in the same scaffold at a different position on the RPW genome as a recent duplicate. We found that an OR (RferSOR1) was the most abundant for both field-collected and lab-reared RPWs, in the rostrum and antennae. Likewise, up-regulated olfactory-related proteins were established in field conditions compared with those from laboratory-reared. We found a rostrum-specific, highly expressing RferSIR1 in IR93a-clade related to hygrosensation. The role of these olfactory proteins as targets for identifying more specific and powerful semiochemicals is discussed in the context of pest management.

Current understandings of olfactory molecular events in the Asian corn borer, Ostrinia furnacalis (Lepidoptera: Crambidae).

The Asian corn borer Ostrinia furnacalis (Lepidoptera: Crambidae) is a serious corn pest with widespread distribution in East Asia. Its olfactory mechanism is a focus of scientific study, aiming to find good ways to control this pest. Molecular events are considered to be important in olfactory mechanism. Current understandings of olfactory molecular events in O. furnacalis, mainly involving sex pheromones and olfactory proteins, were summarized to provide a reference for further studies. O. furnacalis sex pheromone contains two components E-12-tetradecenyl acetate and Z-12-tetradecenyl acetate, which may be recognized and bound by the pheromone binding proteins OfurPBP3 and OfurPBP2, and then transported to the odorant receptors (ORs)OfurOR4 and OfurOR6 to activate them. The ORs OfurOR8, OfurOR7 and OfurOR5b mainly respond to the sex pheromone components of other Ostrinia species, E-11-tetradecenyl acetate, Z-11-tetradecenyl acetate and Z-9-tetradecenyl acetate. The OROfurOR27 responds strongly to plant odorants nonanal, octanal and 1-octanol. Much work remains to be done to fully understand odorants with olfactory activity to O. furnacalis and the functions of its olfactory proteins. These studies will help to reveal olfactory mechanism in O. furnacalis, with the aim of regulating its behaviors to control this pest.

Molecular Characterization and Expression Patterns of Two Pheromone-Binding Proteins from the Diurnal Moth Phauda flammans (Walker) (Lepidoptera: Zygaenoidea: Phaudidae).

Sex pheromone-binding proteins (PBPs) play an important role in sex pheromone recognition in Lepidoptera. However, the mechanisms of chemical communication mediating the response to sex pheromones remain unclear in the diurnal moths of the superfamily Zygaenoidea. In this study, Phauda flammans (Walker) (Lepidoptera: Zygaenoidea: Phaudidae) was used as a model insect to explore the molecular mechanism of sex pheromone perception in the superfamily Zygaenoidea. Two novel pheromone-binding proteins (PflaPBP1 and PflaPBP2) from P. flammans were identified. The two pheromone-binding proteins were predominantly expressed in the antennae of P. flammans male and female moths, in which PflaPBP1 had stronger binding affinity to the female sex pheromones Z-9-hexadecenal and (Z, Z, Z)-9, 12, 15-octadecatrienal, PflaPBP2 had stronger binding affinity only for (Z, Z, Z)-9, 12, 15-octadecatrienal, and no apparent binding affinity to Z-9-hexadecenal. The molecular docking results indicated that Ile 170 and Leu 169 are predicted to be important in the binding of the sex pheromone to PflaPBP1 and PflaPBP2. We concluded that PflaPBP1 and PflaPBP2 may be responsible for the recognition of two sex pheromone components and may function differently in female and male P. flammans. These results provide a foundation for the development of pest control by exploring sex pheromone blocking agents and the application of sex pheromones and their analogs for insect pests in the superfamily Zygaenoidea.

Olfactory Gene Families in Scopula subpunctaria and Candidates for Type-II Sex Pheromone Detection

Scopula subpunctaria, an abundant pest in tea gardens, produce type-II sex pheromone components, which are critical for its communicative and reproductive abilities; however, genes encoding the proteins involved in the detection of type-II sex pheromone components have rarely been documented in moths. In the present study, we sequenced the transcriptomes of the male and female S. subpunctaria antennae. A total of 150 candidate olfaction genes, comprising 58 odorant receptors (SsubORs), 26 ionotropic receptors (SsubIRs), 24 chemosensory proteins (SsubCSPs), 40 odorant-binding proteins (SsubOBPs), and 2 sensory neuron membrane proteins (SsubSNMPs) were identified in S. subpunctaria. Phylogenetic analysis, qPCR, and mRNA abundance analysis results suggested that SsubOR46 may be the Orco (non-traditional odorant receptor, a subfamily of ORs) of S. subpunctaria. SsubOR9, SsubOR53, and SsubOR55 belonged to the pheromone receptor (PR) clades which have a higher expression in male antennae. Interestingly, SsubOR44 was uniquely expressed in the antennae, with a higher expression in males than in females. SsubOBP25, SsubOBP27, and SsubOBP28 were clustered into the moth pheromone-binding protein (PBP) sub-family, and they were uniquely expressed in the antennae, with a higher expression in males than in females. SsubOBP19, a member of the GOBP2 group, was the most abundant OBP in the antennae. These findings indicate that these olfactory genes, comprising five candidate PRs, three candidate PBPs, and one candidate GOBP2, may be involved in type II sex pheromone detection. As well as these genes, most of the remaining SsubORs, and all of the SsubIRs, showed a considerably higher expression in the female antennae than in the male antennae. Many of these, including SsubOR40, SsubOR42, SsubOR43, and SsubIR26, were more abundant in female antennae. These olfactory and ionotropic receptors may be related to the detection of host plant volatiles. The results of this present study provide a basis for exploring the olfaction mechanisms in S. subpunctaria, with a focus on the genes involved in type II sex pheromones. The evolutionary analyses in our study provide new insights into the differentiation and evolution of lepidopteran PRs.

Functional disparity of four pheromone-binding proteins from the plum fruit moth Grapholita funebrana Treitscheke in detection of sex pheromone components

Grapholita funebrana, also known as the plum fruit moth, is an oligophagous pest species that causes enormous economic losses of the fruits of Rosaceae. An eco-friendly method for the control of G. funebrana besides chemical control has not yet been developed. The sex pheromone communication system plays an important role in moth courtship and mating, in which pheromone-binding proteins (PBPs) are critical. In this research, we identified four PBPs, namely, GfunPBP1.1, GfunPBP1.2, GfunPBP2, and GfunPBP3, from the antennae of G. funebrana. The results of real-time quantitative PCR (RT-qPCR) showed that all four GfunPBPs were overwhelmingly expressed in the antennae and that GfunPBP1.2 and GfunPBP2 showed male-biased expression patterns, whereas GfunPBP1.1 and GfunPBP3 were equally expressed between sexes. The results of ligand-binding assays illustrated that although all four recombinant GfunPBPs (rGfunPBPs) had binding activity with the tested sex pheromone compounds, their preferred ligands were significantly different. rGfunPBP2 had the strongest binding affinity to Z8–12:Ac and Z8–12:OH; rGfunPBP1.1 preferred to bind Z8–14:Ac, Z10–14:Ac, and 12:OH more than to the other three GfunPBPs; and rGfunPBP1.2 exhibited stronger binding affinity to E8–12:Ac than to the other rGfunPBPs. Molecular docking results demonstrated that hydrophobic forces, especially van der Waals forces and hydrogen bonds, were the most important forces that maintained GfunPBP–pheromone ligand complexes. This study will improve our understanding of the sex pheromone recognition mechanisms of G. funebrana and promote the development of novel strategies for controlling G. funebrana.

Ostrinia furnacalis PBP2 solution NMR structure: Insight into ligand binding and release mechanisms.

Ostrinia furnacalis is an invasive lepidopteran agricultural pest that relies on olfaction for mating and reproduction. Male moths have an extremely sensitive olfactory system that can detect the sex pheromones emitted by females over a great distance. Pheromone-binding proteins present in the male moth antenna play a key role in the pheromone uptake, transport, and release at the dendritic membrane of the olfactory neuron. Here, we report the first high-resolution NMR structure of a pheromone-binding protein from an Ostrinia species at pH 6.5. The core of the Ostrinia furnacalis PBP2 (OfurPBP2) consists of six helices, α1a (2-14), α1b (16-22), α2 (27-37), α3 (46-60), α4 (70-80), α5 (84-100), and α6 (107-124) surrounding a large hydrophobic pocket. The structure is stabilized by three disulfide bridges, 19-54, 50-108, and 97-117. In contrast to the unstructured C-terminus of other lepidopteran PBPs, the C-terminus of OfurPBP2 folds into an α-helix (α7) at pH 6.5. The protein has nanomolar affinity towards both pheromone isomers. Molecular docking of both pheromones, E-12 and Z-12-tetradecenyl acetate, to OfurPBP2 revealed that the residues Met5, Lys6, Met8, Thr9, Phe12, Phe36, Trp37, Phe76, Ser115, Phe118, Lys119, Ile122, His123, and Ala128 interact with both isomers, while Thr9 formed a hydrogen bond with the acetate head group. NMR structure and thermal unfolding studies with CD suggest that ligand release at pH 4.5 is likely due to the partial unfolding of the protein.

Antennal Transcriptome of the Fruit-Sucking Moth Eudocima materna: Identification of Olfactory Genes and Preliminary Evidence for RNA-Editing Events in Odorant Receptors.

Unappealing shriveled fruits are a characteristic of one of the most elusive fruit pests. The perpetrator, Eudocima materna, attacks the fruit at a fully formed stage and, therefore, the antennal transcriptome for this insect was deduced to identify the molecular elicitors involved in the attraction to its host plants. A total of 260 olfactory genes, including 16 odorant-binding proteins (OBPs), four pheromone-binding proteins (PBPs), 40 antennal-binding proteins (ABPs), 178 odorant receptors (ORs), 17 chemosensory proteins (CSPs) and five sensory neuron membrane proteins (SNMPs) were identified. Phylogenetic analysis shows the divergence of E. materna proteins from closely related lepidopterans and provides insights on genes that have exclusively evolved in this insect. STRING network analysis revealed interactions of olfactory proteins among themselves and the proteins of other groups. Interestingly, online tools predicted RNA-editing events in the odorant receptor sequences, suggesting the possibility of multiple protein forms. Transcripts matching transposable element sequences were also detected in the dataset. Thus, the work reported here provides a valuable resource to design molecular methods for pest control.

The Antennal Sensilla and Expression Patterns of Olfactory Genes in the Lower Termite Reticulitermes aculabialis (Isoptera: Rhinotermitidae).

The insect olfactory system plays pivotal roles in insect survival and reproduction through odor detection. Morphological and physiological adaptations are caste-specific and evolved independently in workers, soldiers, and reproductives in termites. However, it is unclear whether the olfactory system is involved in the division of labor in termite colonies. In the present study, the antennal sensilla of alates, workers, soldiers, nymphs, and larvae of the termite Reticulitermes aculabialis Tsai et Hwang ( Isoptera: Rhinotermitidae) were investigated. Transcriptomes were used to detect olfactory genes, and differential expression levels of olfactory genes were confirmed in various castes by qRT–PCR analysis. Nine types of sensilla were identified on the antennae of R. aculabialis, and soldiers possessed all 9 types. In 89,475 assembled unigenes, we found 16 olfactory genes, including 6 chemosensory protein (CSP) and 10 odorant-binding protein (OBP) genes. These OBP genes included 8 general odorant-binding protein genes (GOBPs) and 2 pheromone-binding protein-related protein (PBP) genes. Five CSP genes were more highly expressed in alates than in workers, soldiers, larvae, and nymphs, and the expression levels of CSP6 were significantly higher in nymphs. Seven GOBP and two PBP genes exhibited significantly higher expression levels in alates, and there were no significant differences in the expression levels of GOBP2 among workers, soldiers, alates, and larvae. These results suggest that alates, as primary reproductives, have unique expression patterns of olfactory genes, which play key roles in nuptial flight, mate seeking, and new colony foundation.

PBP1 plays key roles in sex pheromone reception of the fall armyworm

Spodoptera frugiperda among the China population employs a four-component sex pheromone blend to accelerate male-female allocation and mating behavior. The underlying molecular mechanism has been incompletely elucidated. In the current study, we showed that differences existed between genders toward the four sex pheromone components, including Z9–14:AC, Z7–12:AC, Z9–12:AC, and Z11–16:AC, in terms of electrophysiological responses and behavioral valences. Male adults were significantly more sensitive to all tested compounds than female adults. Furthermore, ecological outputs may be related to four pheromone-binding proteins, namely, SfruPBP1, SfruPBP2, SfruPBP3, and SfruPBP4. They formed four distinct clades within the lepidopteran phylogeny, and male adults expressed significantly higher levels of SfruPBP1 and SfruPBP2 than female adults. We observed the highest binding affinities of SfruPBP1 toward all four sex pheromone components. SfruPBP4 had moderate binding affinities for Z7–12:AC, Z11–16:AC, and Z9–12:AC, while SfruPBP2 showed binding toward Z9–14:AC. This observation suggests that SfruPBP1 plays a key role in sex pheromone discrimination and drives sexually biased behavioral decisions toward certain pheromone components. These findings will help to develop behavioral-mediating tools as part of integrated pest management approaches for this cross-border pest.

Research Progress on the Dendrolimus spp. Pheromone: From Identification to Molecular Recognition.

Dendrolimus species (Lepidoptera, Lasiocampidae), are the most serious phytophagous pests of coniferous forests worldwide. Dendrolimus feed intensively on needles, leading to considerable economic loss and ecological damage. Notably, the outbreak of Dendrolimus is a somewhat periodic pattern, and those outbreaks cause rapid and large-scale destruction of pine forests, with those forests observed to look like “Fire without smoke”. Sex pheromones play an important role during insect mating and reproduction, and there has been extensive research into the pheromone of Dendrolimus. The pheromone components of several Dendrolimus have been identified, and functions of two most important pheromone recognition genes, pheromone-binding proteins (PBPs) and pheromone receptors (PRs), were clarified. The evolution of PBP gene sequences is in good agreement with the trends in structural changes of the sex pheromone components in several Dendrolimus species, and it is interesting that PRs of Dendrolimus spp. occupy a novel lineage of PRs tuned to Type I pheromones in Lepidoptera. We present the current state of research into the sex pheromone of these important forest pests and highlight the emerging topics, to clarify future urgent work into Dendrolimus.