Pheromone-binding Proteins Research Articles

Molecular and Functional Characterization of pheromone binding protein 1 from the Oriental Fruit Moth, Grapholita molesta (Busck)

Pheromone binding protein (PBP) is thought primarily to bind and transport the sex pheromone in moths. The accumulated studies suggest that three PBPs were identified in moth species. In Grapholita molesta, the functions of GmolPBP2 and GmolPBP3 have been previously studied. However, the function of GmolPBP1 is still unclear. Furthermore, the Cydia pomonella sex pheromone Codlemone can act as a sex pheromone synergist of G. molesta. In C. pomonella, CpomPBP1 specifically bind the Codlemone. CpomPBP1 displays high identity with GmolPBP1 (70%), indicating that the two PBPs may share a similar 3D structure thus can bind the similar or same ligands. In this study, we explored the molecular and functional characterization of GmolPBP1. GmolPBP1, bearing the typical characteristics of Lepidopteran odorant binding proteins, was closest phylogenetically to CpomPBP1. Binding studies demonstrated that GmolPBP1 exhibited strong binding affinities with (Z)-8-dodecenyl alcohol, 1-dodecanol and Codlemone. Molecular docking showed that GmolPBP1 has different ligand recognition mechanism for the three ligands. Our results suggest that GmolPBP1 functions as recognizer of (Z)-8-dodecenyl alcohol and 1-dodecanol of the female sex pheromone blend, and may be the potential transporter of Codlemone, which contributes to the synergism of the pheromone response of G. molesta by Codlemone.

Correlation of pheromone-binding protein–ligand equilibrium dissociation constants with electroantennogram response patterns

Pheromone-binding proteins (PBPs) are water-soluble proteins found at high concentration in the lymph fluid of pheromone-sensing hairs on insect antennae. PBPs could function as pheromone transporters, ferrying the hydrophobic odorants to their cognate odorant receptors. However, it is also possible for these proteins to bind the odorants near the dendritic membrane of pheromone-sensing neurons and, therefore, function as scavengers. The two functions are not mutually exclusive. In this paper, the transporter and (or) scavenger roles of PBPs in pheromone perception were investigated using the pheromone of the gypsy moth (7R, 8S)-epoxy-2-methyloctadecane and analogues with heteroatom (O or S) substitutions in the hydrocarbon chain. PBP–ligand equilibrium dissociation constants (Kd) were correlated with electroantennogram (EAG) response patterns of male gypsy moth antennae to the pheromone, its enantiomer, and their respective analogues. EAG measures the potential drop across the antenna due to odorant receptor activation and subsequent ion channel opening. Three quantifiable properties of the EAG responses were used: lag times from stimulus to response onset, depolarization rates (rate of receptor activation), and repolarization rates (rate of receptor deactivation). Negative correlations were observed between Kd and lag times and between Kd and repolarization rates. Positive correlations were seen with Kd against depolarization rates. The inverse relationship of Kd constants with lag times and the direct relationship with depolarization rates strongly supports transporter function of PBPs. Interestingly, the inverse correlation of Kd constants with repolarization rates suggests a scavenger effect. These results indicate that PBP affects odorant receptor activity through both odorant transport and scavenger functions. Through differences in ligand binding affinities, PBPs influence pheromone availability for receptor activation.

Identification of pheromone-binding proteins of the maize stem borer, Chilo partellus (Swinhoe, 1885) (Lepidoptera: Crambidae)

Pheromone-binding proteins (PBPs) play a significant role in olfaction and mating. The present work was designed to isolate, extract, and purify the pheromone-binding proteins from the antennae of male Chilo partellus (Swinhoe, 1885) (Lepidoptera: Crambidae). The pheromone-binding proteins extracted from the male antennae were found to be 770 μg in 100 mg of sample. Pheromone-binding protein molecular weight was determined as 17 kDa by SDS-PAGE analysis. Identified proteins were purified through gel extraction with a recovery percentage of proteins up to 95%. Purified protein samples are analyzed on native PAGE gels. Relative mobility of proteins was determined as 0.574 nm in the densitometry analysis. These identified pheromone-binding proteins can be used for identification of novel pheromone compounds in in vitro studies. This study can be helpful in designing integrated pest management programs to control the maize stem borer by mass trapping of male moths.

Differential gene expression in the evolution of sex pheromone communication in New Zealand\u2019s endemic leafroller moths of the genera Ctenopseustis and Planotortrix

BackgroundSex pheromone communication in moths has attracted the attention of evolutionary biologists due to the vast array of pheromone compounds used, addressing questions of how this diversity arose and how male reception has evolved in step with the female signal. Here we examine the role of changing gene expression in the evolution of mate recognition systems in leafroller moths, particularly focusing on genes involved in the biosynthetic pathways of sex pheromones in female pheromone glands and the peripheral reception repertoire in the antennae of males. From tissue-specific transcriptomes we mined and compared a database of genes expressed in the pheromone glands and antennae of males and females of four closely related species of leafroller moths endemic to New Zealand, Ctenopseutis herana and C. obliquana, and Planotortrix excessana and P. octo. The peculiarity of this group, compared to other Lepidoptera, is the use of (Z)-5-tetradecenyl acetate, (Z)-7-tetradecenyl acetate, and (Z)-8-tetradecenyl acetate as sex pheromone components.ResultsWe identify orthologues of candidate genes from the pheromone biosynthesis pathway, degradation and transport, as well as genes of the periphery olfactory repertoire, including large families of binding proteins, receptors and odorant degrading enzymes. The production of distinct pheromone blends in the sibling species is associated with the differential expression of two desaturase genes, deast5 and desat7, in the pheromone glands. In male antennae, three odorant receptors, OR74, OR76a and OR30 are over-expressed, but their expression could not be clearly associated with the detection of species-specific pheromones components. In addition these species contain duplications of all three pheromone binding proteins (PBPs) that are also differentially expressed among species.ConclusionsWhile in females differences in the expression of desaturases may be sufficient to explain pheromone blend differences among these New Zealand leafroller species, in males differential expression of several genes, including pheromone binding proteins, may underpin differences in the response by males to changing pheromone components among the species.

CRISPR/Cas9-mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis.

Pheromone‐binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo. In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP‐single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target‐gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBP1‐sgRNA injected eggs and 19.5% for PBP3‐sgRNA injected eggs. Second, by an in‐crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened, and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA‐injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.

Key site residues of pheromone-binding protein 1 involved in interacting with sex pheromone components of Helicoverpa armigera

Pheromone binding proteins (PBPs) are widely distributed in insect antennae, and play important roles in the perception of sex pheromones. However, the detail mechanism of interaction between PBPs and odorants remains in a black box. Here, a predicted 3D structure of PBP1 of the serious agricultural pest, Helicoverpa armigera (HarmPBP1) was constructed, and the key residues that contribute to binding with the major sex pheromone components of this pest, (Z)-11- hexadecenal (Z11-16:Ald) and (Z)-9- hexadecenal (Z9-16:Ald), were predicted by molecular docking. The results of molecular simulation suggest that hydrophobic interactions are the main linkage between HarmPBP1 and the two aldehydes, and four residues in the binding pocket (Phe12, Phe36, Trp37, and Phe119) may participate in binding with these two ligands. Then site-directed mutagenesis and fluorescence binding assays were performed, and significant decrease of the binding ability to both Z11-16:Ald and Z9-16:Ald was observed in three mutants of HarmPBP1 (F12A, W37A, and F119A). These results revealed that Phe12, Trp37, and Phe119 are the key residues of HarmPBP1 in binding with the Z11-16:Ald and Z9-16:Ald. This study provides new insights into the interactions between pheromone and PBP, and may serve as a foundation for better understanding of the pheromone recognition in moths.

Identification of the pheromone biosynthesis genes from the sex pheromone gland transcriptome of the diamondback moth, Plutella xylostella

The diamondback moth was estimated to increase costs to the global agricultural economy as the global area increase of Brassica vegetable crops and oilseed rape. Sex pheromones traps are outstanding tools available in Integrated Pest Management for many years and provides an effective approach for DBM population monitoring and control. The ratio of two major sex pheromone compounds shows geographical variations. However, the limitation of our information in the DBM pheromone biosynthesis dampens our understanding of the ratio diversity of pheromone compounds. Here, we constructed a transcriptomic library from the DBM pheromone gland and identified genes putatively involved in the fatty acid biosynthesis, pheromones functional group transfer, and β-oxidation enzymes. In addition, odorant binding protein, chemosensory protein and pheromone binding protein genes encoded in the pheromone gland transcriptome, suggest that female DBM moths may receive odors or pheromone compounds via their pheromone gland and ovipositor system. Tissue expression profiles further revealed that two ALR, three DES and one FAR5 genes were pheromone gland tissue biased, while some chemoreception genes expressed extensively in PG, pupa, antenna and legs tissues. Finally, the candidate genes from large-scale transcriptome information may be useful for characterizing a presumed biosynthetic pathway of the DBM sex pheromone.

Chemosensory Gene Families in Ectropis grisescens and Candidates for Detection of Type-II Sex Pheromones.

Tea grey geometrid (Ectropis grisescens), a devastating chewing pest in tea plantations throughout China, produces Type-II pheromone components. Little is known about the genes encoding proteins involved in the perception of Type-II sex pheromone components. To investigate the olfaction genes involved in E. grisescens sex pheromones and plant volatiles perception, we sequenced female and male antennae transcriptomes of E. grisescens. After assembly and annotation, we identified 153 candidate chemoreception genes in E. grisescens, including 40 odorant-binding proteins (OBPs), 30 chemosensory proteins (CSPs), 59 odorant receptors (ORs), and 24 ionotropic receptors (IRs). The results of phylogenetic, qPCR, and mRNA abundance analyses suggested that three candidate pheromone-binding proteins (EgriOBP2, 3, and 25), two candidate general odorant-binding proteins (EgriOBP1 and 29), six pheromone receptors (EgriOR24, 25, 28, 31, 37, and 44), and EgriCSP8 may be involved in the detection of Type-II sex pheromone components. Functional investigation by heterologous expression in Xenopus oocytes revealed that EgriOR31 was robustly tuned to the E. grisescens sex pheromone component (Z,Z,Z)-3,6,9-octadecatriene and weakly to the other sex pheromone component (Z,Z)-3,9-6,7-epoxyoctadecadiene. Our results represent a systematic functional analysis of the molecular mechanism of olfaction perception in E. grisescens with an emphasis on gene encoding proteins involved in perception of Type-II sex pheromones, and provide information that will be relevant to other Lepidoptera species.

Functional characterization of PBP1 gene in Helicoverpa armigera (Lepidoptera: Noctuidae) by using the CRISPR/Cas9 system

Pheromone binding proteins (PBPs) are thought to play crucial roles in perception of the sex pheromones particularly in noctuid moths, but this is rarely in vivo evidenced due to lacking an effective technique. Here, we reported an in vivo functional study of PBP1 in the important lepidopteran pest Helicoverpa armigera (HarmPBP1), by using the CRISPR/Cas9 system. Efficient and heritable mutagenesis was achieved by egg injection of mixture of Cas9-mRNA and HarmPBP1-sgRNA. The TA cloning and sequencing revealed various insertion and/or deletion (indel) mutations at the target site. Among those, one mutation resulted in a premature stop codon at the target site, which led to a highly truncated protein with only 10 amino acids. The HarmPBP1 with this mutation would completely loss its function, and thus was used to select the homozygous mutant insects for functional analysis. The electroantennogram recording showed that the mutant male adults displayed severely impaired responses to all three sex pheromone components (Z11-16:Ald, Z9-16:Ald and Z9-14:Ald). Our study provides the first in vivo evidence that HarmPBP1 plays important role in perception of female sex pheromones, and also an effective methodology for using CRISPR/Cas9 system in functional genetic study in H. armigera as well as other insects.

Tissue distribution and functional characterization of odorant binding proteins in Chilo suppressalis (Lepidoptera: Pyralidae)

Odorant binding proteins (OBPs) play important roles in the insect olfaction and other diverse physiological processes. Forty OBP genes have been molecularly identified from Chilo suppressalis (Walker), a notorious rice pest in Asian countries, but little is known about the olfactory function for most of these genes. In the present study, we first determined the tissue expression profiles of 34 OBPs (excluding two general odorant bonding proteins (GOBPs) and four pheromone binding protein (PBPs)) by quantitative real-time PCR (qPCR), and found that 9 genes (OBP1, 3, 4, 11, 15, 17, 19, 20 and 24) were specifically or predominantly expressed in antennae of both sexes, suggesting their roles in olfaction, while three genes (OBP29, 30 and 32) were almost not expressed in antennae. Focusing on olfactory roles, the ligand specificities of six antenna specifically or predominantly expressed genes were further investigated for 35 plant volatiles, using the fluorescence competitive binding assays. The results revealed that six OBPs displayed different ligand preference, suggesting a differentiation of OBPs in ligand binding spectrum. Of six tested OBPs, OBP3, 11, 17, 19 and 31 showed moderate (Ki=10.21–19.85μM) or high (Ki<10.00μM) binding affinity for 11 and one plant volatiles, respectively. In particular, a plant volatile β-ionone had high or moderate binding to all five OBPs. Our study suggests that these five OBP genes play important roles in the perception of different host plant volatiles, providing insight into the olfactory mechanism in C. suppressalis.

Identification of odorant-binding and chemosensory protein genes and the ligand affinity of two of the encoded proteins suggest a complex olfactory perception system in Periplaneta americana.

The American cockroach (Periplaneta americana) is an urban pest with a precise chemosensory system that helps it achieve complex physiological behaviours, including locating food and mating. However, its chemosensory mechanisms have not been well studied. Here, we identified 71 putative odorant carrier protein genes in P.americana, including 57 new odorant-binding proteins (OBPs) and 11 chemosensory proteins (CSPs). To identify their physiological functions, we investigated their tissue expression patterns in antennae, mouthparts, legs, and the remainder of the body of both sexes, and determined that most of these genes were expressed in chemosensory organs. A phylogenetic tree showed that the putative pheromone-binding proteins of P.americana were in different clades from those of moths. Two genes, PameOBP24 and PameCSP7, were expressed equally in antennae of both sexes and highly expressed amongst the OBPs and CSPs. These genes were expressed in Escherichia coli and the resultant proteins were purified. The binding affinities of 74 common odorant compounds were tested with recombinant PameOBP24 and PameCSP7. Both proteins bound a variety of ligands. Our findings provide a foundation for future research into the chemosensory mechanisms of P.americana and help in identifying potential target genes for managing this pest.

Identification and Expression Profiling of Chemosensory Genes in Dendrolimus punctatus Walker

Dendrolimus punctatus Walker is a serious pest affecting conifers in southern China. As extensive pesticide spraying is currently required to control D. punctatus, new control strategies are urgently needed. Chemosensory genes represent potential molecular targets for development of alternative pest control strategies, and the expression characteristics of these genes provide an indication of their function. To date, little information is available regarding chemosensory genes in D. punctatus or their expression profiles at different development stages and in various tissues. Here, we assembled and analyzed the transcriptomes of D. punctatus collected at different developmental stages and in a range of organs, using next-generation sequencing. A total of 171 putative chemosensory genes were identified, encoding 53 odorant binding proteins, 26 chemosensory proteins, 60 odorant receptors (OR), 12 gustatory receptors (GR), 18 ionotropic receptors (IR), and 2 sensory neuron membrane proteins (SNMPs). Expression analysis indicated that the antennae possess the largest number of highly expressed olfactory genes and that olfactory gene expression patterns in the eggs, larvae, and head were similar to one another, with each having moderate numbers of highly expressed olfactory genes. Fat body, ovary, midgut, and testis tissues also had similar olfactory gene expression patterns, including few highly expressed olfactory genes. Of particular note, we identified only two pheromone binding proteins and no pheromone receptors in D. punctatus, similar to our previous findings in Dendrolimus houi and Dendrolimus kikuchii, suggesting that insects of the Dendrolimus genus have different pheromone recognition characteristics to other Lepidopteran insects. Overall, this extensive expression profile analysis provides a clear map of D. punctatus chemosensory genes, and will facilitate functional studies and the development of new pest control methods in the future.

The developmental transcriptome of the bamboo snout beetle Cyrtotrachelus buqueti and insights into candidate pheromone-binding proteins.

Cyrtotrachelus buqueti is an extremely harmful bamboo borer, and the larvae of this pest attack clumping bamboo shoots. Pheromone-binding proteins (PBPs) play an important role in identifying insect sex pheromones, but the C. buqueti genome is not readily available for PBP analysis. Developmental transcriptomes of eggs, larvae from the first instar to the prepupal stage, pupae, and adults (females and males) from emergence to mating were built by RNA sequencing (RNA-Seq) in the present study to establish a sequence background of C. buqueti to help understand PBPs. Approximately 164.8 million clean reads were obtained and annotated into 108,854 transcripts. These were assembled into 24,338, 21,597, 24,798, 21,886, 24,642, and 83,115 unigenes for eggs, larvae, pupae, females, males, and the combined datasets, respectively. Unigenes were annotated against NCBI non-redundant protein sequences, NCBI non-redundant nucleotide sequences, Gene Ontology (GO), Protein family, Clusters of Orthologous Groups of Proteins/ Clusters of Eukaryotic Orthologous Groups (KOG), Swiss-Prot, and KEGG Orthology databases. A total of 17,213 unigenes were annotated into 55 sub-categories belonging to three main GO categories; 10,672 unigenes were classified into 26 functional categories by KOG classification, and 8,063 unigenes were classified into five functional KEGG categories. RSEM software for RNA sequencing showed that 4,816, 3,176, 3,661, 2,898, 4,316, 8,019, 7,273, 5,922, 5,844, and 4,570 genes were differentially expressed between larvae and males, larvae and eggs, larvae and pupae, larvae and females, males and females, males and eggs, males and pupae, females and eggs, females and pupae, and eggs and pupae, respectively. Of these, three were confirmed to be significantly differentially expressed between larvae, females, and males. Furthermore, PBP Cbuq7577_g1 was highly expressed in the antenna of males. A comprehensive sequence resource of a desirable quality was constructed from developmental transcriptomes of C. buqueti eggs, larvae, pupae, and adults. This work enriches the genomic data of C. buqueti, and facilitates our understanding of its metamorphosis, development, and response to environmental change. The identified candidate PBP Cbuq7577_g1 might play a crucial role in identifying sex pheromones, and could be used as a targeted gene to control C. buqueti numbers by disrupting sex pheromone communication.

Antennal transcriptome analysis of the piercing moth Oraesia emarginata (Lepidoptera: Noctuidae).

The piercing fruit moth Oraesia emarginata is an economically significant pest; however, our understanding of its olfactory mechanisms in infestation is limited. The present study conducted antennal transcriptome analysis of olfactory genes using real-time quantitative reverse transcription PCR analysis (RT-qPCR). We identified a total of 104 candidate chemosensory genes from several gene families, including 35 olfactory receptors (ORs), 41 odorant-binding proteins, 20 chemosensory proteins, 6 ionotropic receptors, and 2 sensory neuron membrane proteins. Seven candidate pheromone receptors (PRs) and 3 candidate pheromone-binding proteins (PBPs) for sex pheromone recognition were found. OemaOR29 and OemaPBP1 had the highest fragments per kb per million fragments (FPKM) values in all ORs and OBPs, respectively. Eighteen olfactory genes were upregulated in females, including 5 candidate PRs, and 20 olfactory genes were upregulated in males, including 2 candidate PRs (OemaOR29 and 4) and 2 PBPs (OemaPBP1 and 3). These genes may have roles in mediating sex-specific behaviors. Most candidate olfactory genes of sex pheromone recognition (except OemaOR29 and OemaPBP3) in O. emarginata were not clustered with those of studied noctuid species (type I pheromone). In addition, OemaOR29 was belonged to cluster PRIII, which comprise proteins that recognize type II pheromones instead of type I pheromones. The structure and function of olfactory genes that encode sex pheromones in O. emarginata might thus differ from those of other studied noctuids. The findings of the present study may help explain the molecular mechanism underlying olfaction and the evolution of olfactory genes encoding sex pheromones in O. emarginata.

Identification of odorant binding proteins in Carpomya vesuviana and their binding affinity to the male-borne semiochemicals and host plant volatiles

Carpomya vesuviana (Costa; Diptera: Tephritidae) is an agricultural pest that causes serious damage to jujube fruits. However, the mechanism of olfaction, which is critical for host identification, is not well understood in this pest. In this study, we have identified for the first time five protein types involved in the olfactory signal transduction of C. vesuviana by using transcriptome sequencing. These include 6 odorant-binding proteins (OBPs), 15 odorant receptors (ORs), 22 gustatory receptors (GRs), 2 chemosensory proteins (CSPs), and 2 sensory neuron membrane proteins (SNMPs). Amino acids alignment and phylogenetic analysis showed that all 6 OBPs have a signal peptide at their respective N-termini with four OBPs belonging with the classic OBPs, and OBP2 and OBP5 belonging to the Minus-C family. OBP3 clustered with the OBP83a/83b clade, which comprised pheromone binding protein related proteins (PBPRPs). Moreover, volatiles from C. vesuviana adults and its host plants were collected and identified by using solid phase microextraction (SPME) and gas-chromatography/mass spectrometry (GC/MS). The results indicated that male adults emitted nonanal, and five other compounds, caryophyllene, chamigrene, camphene, (Z)-3-hexen-1-ol acetate, and ocimene were identified in the fruits of jujubes. Electroantennogram (EAG) assays revealed that adult C. vesuviana responded to all six compounds along with two additional pheromones (geranyl acetate and α-farnesene) from other tephritids and the values ranged from 0.50mV to 1.26mV. To further explore the interaction between OBPs and volatiles, competitive binding assays were carried out. The results showed that only CvesOBP2 had binding affinity to (Z)-3-hexen-1-ol acetate. OBP5 and OBP6 exhibited broad spectrum binding to compounds with relatively low molecular weights, and OBP1 and OBP4 had some affinity to caryophyllene and chamigrene. However, OBP3 exhibited relatively high binding affinity to α-farnesene. The findings of this study provide insights into the olfactory mechanisms and the potential functions of OBPs in the olfactory reception pathway in C. vesuviana. The OBPs identified in this study could be used as potential targets to develop attractants to monitor this insect pest for effective pest control.

Identification of candidate chemosensory genes by transcriptome analysis in Loxostege sticticalis Linnaeus.

Loxostege sticticalis Linnaeus is an economically important agricultural pest, and the larvae cause great damage to crops, especially in Northern China. However, effective and environmentally friendly chemical methods for controlling this pest have not been discovered to date. In the present study, we performed HiSeq2500 sequencing of transcriptomes of the male and female adult antennae, adult legs and third instar larvae, and we identified 54 candidate odorant receptors (ORs), including 1 odorant receptor coreceptor (Orco) and 5 pheromone receptors (PRs), 18 ionotropic receptors (IRs), 13 gustatory receptors (GRs), 34 odorant binding proteins (OBPs), including 1 general odorant binding protein (GOBP1) and 3 pheromone binding proteins (PBPs), 10 chemosensory proteins (CSPs) and 2 sensory neuron membrane proteins (SNMPs). The results of RNA-Seq and RT-qPCR analyses showed the expression levels of most genes in the antennae were higher than that in the legs and larvae. Furthermore, PR4, OR1-4, 7–11, 13–15, 23, 29–32, 34, 41, 43, 47/IR7d.2/GR5b, 45, 7/PBP2-3, GOBP1, OBP3, 8 showed female antennae-biased expression, while PR1/OBP2, 7/IR75d/CSP2 showed male antennae-biased expression. However, IR1, 7d.3, 68a/OBP11, 20–22, 28/CSP9 had larvae enriched expression, and OBP15, 17, 25, 29/CSP5 were mainly expressed in the legs. The results shown above indicated that these genes might play a key role in foraging, seeking mates and host recognition in the L. sticticalis. Our findings will provide the basic knowledge for further studies on the molecular mechanisms of the olfactory system of L. sticticalis and potential novel targets for pest control strategies.

Molecular elements of pheromone detection in the female moth, Heliothis virescens.

Pheromones play pivotal roles in the reproductive behavior of moths, most prominently for the mate finding of male moths. Accordingly, the molecular basis for the detection of female-released pheromones by male moths has been studied in great detail. In contrast, little is known about how females can detect pheromone components released by themselves or by conspecifics. In this study, we assessed the antenna of female Heliothis virescens for elements of pheromone detection. In accordance with previous findings that female antennae respond to the sex pheromone component (Z)-9-tetradecenal, we identified olfactory sensory neurons that express its cognate receptor, the receptor type HR6. All HR6 cells coexpressed the "sensory neuron membrane protein 1" (SNMP1) and were associated with supporting cells expressing the pheromone-binding proteins PBP1 and PBP2. These features are reminiscent to male antennae and point to congruent mechanisms for pheromone detection in the two sexes. Further analysis of the SNMP1-expressing cells revealed a higher number in females compared to males. Moreover, in females, the SNMP1 neurons were arranged in clusters, which project their dendrites into a common sensillum, whereas in males there were only solitary SNMP1-neurons and only 1 per sensillum. Not all SNMP1 positive cells in female antennae expressed HR6 but instead the putative pheromone receptors HR11 and HR18, respectively. Neurons expressing 1 of the 3 receptor types were assigned to different sensilla. Together the data indicate that on the antenna of females, sensory neurons in a subset of sensilla trichodea are equipped with molecular elements, which render them responsive to pheromones.

Comparative Transcriptome Analysis of ThreeBactrocera dorsalis(Diptera: Tephritidae) Organs to Identify Functional Genes in the Male Accessory Glands and Ejaculatory Duct

The insect male accessory glands/ejaculatory duct (MAG/ED) are important tissues of the male reproductive system. The MAG/ED's functions in reproduction have been well studied in Drosophila (Diptera: Drosophilidae) but remain largely unknown in the important agricultural pest Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). In the present study, we re-assembled the transcriptome datasets of B. dorsalis's fat body, testis, and MAG/ED and compared these tissue-specific transcriptomes. Clean reads from these transcriptome data sets were de novo re-assembled and clustered into 31,782 unigenes (average 922 bp). In total, 21,306 unigenes were functionally annotated by Blasting against online databases. Comparative transcriptomic analysis identified numerous genes that were identified with the expressed tissue-bias patterns. Some MAG/ED-specific genes potentially involved in spermatozoa motility and capacitation (e.g., perlucin, glucose dehydrogenase, lipase), mating regulation (pheromone-binding protein—related protein), and immunity (lectin) were identified in B. dorsalis. The expressions of some of these genes were further validated by realtime quantitative polymerase chain reaction at transcriptional level. All of these identifications will help us to explore the physiological regulation of mating and reproduction in B. dorsalis in the future.

RNAi-Induced Electrophysiological and Behavioral Changes Reveal two Pheromone Binding Proteins of Helicoverpa armigera Involved in the Perception of the Main Sex Pheromone Component Z11-16:Ald.

Pheromone binding proteins (PBPs) are thought to play key roles in insect sex pheromone recognition; however, there is little in vivo evidence to support this viewpoint in comparison to abundant biochemical data in vitro. In the present study, two noctuid PBP genes HarmPBP1 and HarmPBP2 of the serious agricultural pest, Helicoverpa armigera were selected to be knocked down by RNA interference, and then the changes in electrophysiological and behavioral responses of male mutants to their major sex pheromone component (Z)-11-hexadecenal (Z11-16:Ald) were recorded. There were no significant electrophysiological or behavioral changes of tested male moths in response to Z11-16:Ald when either single PBP gene was knocked down. However, decreased sensitivity of male moths in response to Z11-16:Ald was observed when both HarmPBP1 and HarmPBP2 genes were silenced. These results reveal that both HarmPBP1 and HarmPBP2 are required for the recognition of the main sex pheromone component Z11-16:Ald in H. armigera. Furthermore, these findings may help clarify physiological roles of moth PBPs in the sex pheromone recognition pathway, which in turn could facilitate pest control by exploring sex pheromone blocking agents.

Structure-based discovery of potentially active semiochemicals for Cydia pomonella (L.).

The development of physiologically active semiochemicals is largely limited by the labor-consuming searching process. How to screen active semiochemicals efficiently is of significance to the extension of behavior regulation in pest control. Here pharmacophore modeling and shape-based virtual screening were combined to predict candidate ligands for Cydia pomonella pheromone binding protein 1 (CpomPBP1). Out of the predicted compounds, ETrME displayed the highest affinity to CpomPBP1. Further studies on the interaction between CpomPBP1 and ETrME, not only depicted the binding mode, but also revealed residues providing negative and positive contributions to the ETrME binding. Moreover, key residues involved in interacting with ETrME of CpomPBP1 were determined as well. These findings were significant to providing insights for the future searching and optimization of active semiochemicals.