Structural basis of the honey bee PBP pheromone and pH-induced conformational change

The behavior of insects and their perception of their surroundings are driven, in a large part, by odorants and pheromones. This is especially true for social insects, such as the honey bee, where the queen controls the development and the caste status of the other individuals. Pheromone perception is a complex phenomenon relying on a cascade of recognition events, initiated in antennae by pheromone recognition by a pheromone-binding protein and finishing with signal transduction at the axon membrane level. With to the objective of deciphering this initial step, we have determined the structures of the bee antennal pheromone-binding protein (ASP1) in the apo form and in complex with the main component of the queen mandibular pheromonal mixture, 9-keto-2(E)-decenoic acid (9-ODA) and with nonpheromonal components. In the apo protein, the C terminus obstructs the binding site. In contrast, ASP1 complexes have different open conformations, depending on the ligand shape, leading to different volumes of the binding cavity. The binding site integrity depends on the C terminus (111-119) conformation, which involves the interplay of two factors; i.e. the presence of a ligand and a low pH. Ligand binding to ASP1 is favored by low pH, opposite to what is observed with other pheromone-binding proteins, such as those of Bombyx mori and Anopheles gambiae.     

Binding properties of pheromone-binding protein 1 from the common cutworm Spodoptera litura

Pheromone-binding proteins (PBPs) were formerly thought to act as passive pheromone carriers. However, recent studies, particularly in Drosophila melanogaster, suggest that PBPs are involved in the recognition of semiochemicals, thus making ligand-binding studies more meaningful. Previously, we cloned three PBPs from Spodoptera litura (Slit), and showed that SlitPBP1 is much more abundant than the other two, particularly in male antennae. To investigate the ligand specificity of SlitPBP1, we expressed the protein in a bacterial system and performed binding experiments with the three components of the specific sex pheromones (Z9-14:Ac, Z9,E11-14:Ac and Z9,E12-14:Ac), as well as with 26 volatile ligands. The results indicated that SlitPBP1 bound all three sex pheromone components with dissociation constants between 0.6 and 1.1 μM. The same protein also bound with comparable affinities several pheromone analogs, but not plant volatiles. The presence of a double bond was the most important element for a strong binding, while its position and configuration also affected the affinity. Finally, the binding of pheromone components is strongly affected by pH, showing a critical pH value corresponding to isoelectric point of the protein. This suggests that a pH-dependent conformational mechanism might exist in SlitPBP1 for pheromone binding and release.     

Identification and Characterization of Pheromone Receptors and Interplay between Receptors and Pheromone Binding Proteins in the Diamondback Moth,Plutella xyllostella

Moths depend on olfactory cues such as sex pheromones to find and recognize mating partners. Pheromone receptors (PRs) and Pheromone binding proteins (PBPs) are thought to be associated with olfactory signal transduction of pheromonal compounds in peripheral olfactory reception. Here six candidate pheromone receptor genes in the diamondback moth, Plutella xyllostella were identified and cloned. All of the six candidate PR genes display male-biased expression, which is a typical characteristic of pheromone receptors. In the Xenopus-based functional study and in situ hybridization, PxylOR4 is defined as another pheromone receptor in addition to the previously characterized PxylOR1. In the study of interaction between PRs and PBPs, PxylPBPs could increase the sensitivity of the complex expressing oocyte cells to the ligand pheromone component while decreasing the sensitivity to pheromone analogs. We deduce that activating pheromone receptors in olfactory receptor neurons requires some role of PBPs to pheromone/PBP complex. If the chemical signal is not the pheromone component, but instead, a pheromone analog with a similar structure, the complex would have a decreased ability to activate downstream pheromone receptors.     

A pheromone-binding protein mediates the bombykol-induced activation of a pheromone receptor in vitro

The enormous capacity of the male silkmoth Bombyx mori in recognizing and discriminating bombykol and bombykal is based on distinct sensory neurons in the antennal sensilla hairs. The hydrophobic pheromonal compounds are supposed to be ferried by soluble pheromone-binding proteins (PBPs) through the sensillum lymph toward the receptors in the dendritic membrane. We have generated stable cell lines expressing the candidate pheromone receptors of B. mori, BmOR-1 or BmOR-3, and assessed their responses to hydrophobic pheromone compounds dissolved by means of dimethyl sulfoxide. BmOR-1-expressing cells were activated by bombykol but also responded to bombykal, whereas cells expressing BmOR-3 responded to bombykal only. In experiments employing the B. mori PBP, no organic solvent was necessary to mediate an activation of BmOR-1 by bombykol, indicating that the PBP solubilizes the hydrophobic compound. Furthermore, the employed PBP selectively mediated a response to bombykol but not to bombykal, supporting a ligand specificity of PBPs. This study provides evidence that both distinct pheromone receptors and PBPs play an important role in insect pheromone recognition.     

Candidate pheromone binding proteins of the silkmoth Bombyx mori

Pheromone reception is thought to be mediated by pheromone binding proteins (PBPs) in the aqueous lymph of the antennal sensilla. Recent studies have shown that the only known PBP of Bombyx mori (BmorPBP1) appears to be specifically tuned to bombykol but not to bombykal, raising the question of whether additional subtypes may exist. We have identified two novel genes, which encode candidate PBPs (BmorPBP2, BmorPBP3). Comparison with PBPs from various moth species have revealed a high degree of sequence identity and the three BmorPBP-subtypes can be assigned to distinct groups within the moth PBP family. In situ hybridization revealed that BmorPBP2 and BmorPBP3 are expressed only in relatively few cells compared to the number of cells expressing BmorPBP1. Double-labeling experiments have shown that the two novel BmorPBPs are expressed in the same cells but are not co-expressed with BmorPBP1. Furthermore, unlike BmorPBP1, cells expressing the newly identified PBPs did not surround neurons containing the BmOR-1 receptor. The results indicate that BmorPBP2 and BmorPBP3 are located in sensilla types, which are different from the long sensilla trichodea.Data deposition: The sequences reported in this paper have been deposited in the EMBL database under accession nos. AM403100 (BmorPBP2) and AM403101 (BmorPBP3).     

Structural and functional difference of pheromone binding proteins in discriminating chemicals in the gypsy moth, Lymantria dispar

Pheromone-binding proteins (PBPs) of the gypsy moth, Lymantria dispar L., play an important role in olfaction. Here structures of PBPs were first built by Homology Modeling, and each model of PBPs had seven α-helices and a large hydrophobic cavity including 25 residues for PBP1 and 30 residues for PBP2. Three potential semiochemicals were first screened by CDOCKER program based on the PBP models and chemical database. These chemicals were Palmitic acid n-butyl ester (Pal), Bis(3,4-epoxycyclohexylmethyl) adipate (Bis), L-trans-epoxysuccinyl-isoleucyl-proline methyl ester propylamide (CA-074). The analysis of chemicals docking the proteins showed one hydrogen bond was established between the residues Lys94 and (+)-Disparlure ((+)-D), and л-л interactions were present between Phe36 of PBP1 and (+)-D. The Lys94 of PBP1 formed two and three hydrogen bonds with Bis and CA-074, respectively. There was no residue of PBP2 interacting with these four chemicals except Bis forming one hydrogen bond with Lys121. After simulating the conformational changes of LdisPBPs at pH7.3 and 5.5 by constant pH molecular dynamics simulation in implicit solvent, the N-terminal sequences of PBPs was unfolded, only having five α-helices, and PBP2 had larger binding pocket at 7.3 than PBP1. To investigate the changes of α-helices at different pH, far-UV and near-UV circular dichroism showed PBPs consist of α-helices, and the tertiary structures of PBP1 and PBP2 were influenced at pH7.3 and 5.5. The fluorescence binding assay indicated that PBP1 and PBP2 have similarly binding affinity to (+)-D at pH 5.5 and 7.3, respectively. At pH 5.5, the dissociation constant of the complex between PBP1 and 2-decyl-1-oxaspiro [2.2] pentane (OXP1) was 0.68 ± 0.01 μM, for (+)-D was 5.32 ± 0.11 μM, while PBP2 with OXP1 and (+)-D were 1.88 ± 0.02 μM and 5.54 ± 0.04 μM, respectively. Three chemicals screened had higher affinity to PBP1 than (+)-D except Pal at pH5.5, and had lower affinity than (+)-D at pH7.3. To PBP2, these chemicals had lower affinity than the sex pheromone except Bis at pH 5.5 and pH 7.3. Only PBP1 had higher affinity with Sal than the sex pheromone at pH 5.5. Therefore, the structures of PBP1 and PBP2 had different changes at pH5.5 and 7.3, showing different affinity to chemicals. This study helps understanding the role of PBPs as well as in developing more efficient chemicals for pest control.     

Structure-activity studies with pheromone-binding proteins of the gypsy moth,Lymantria dispar

Pheromone olfaction in the gypsy moth, Lymantria dispar, involves accurate distinction of compounds with similar structure and polarity. The identified sex pheromone is (7R,8S)-2-methyl-7,8-epoxyoctadecane, 1a, and a known antagonist is (7Z)-2-methyloctadec-7-ene, 4a. The first step in pheromone olfaction is binding of odorants by small, soluble pheromone-binding proteins (PBPs), found in the pheromone-sensing hairs. We have studied the molecular determinants recognized by the two PBPs found in the gypsy moth, using three pheromone/PBP binding assays. Results indicate that (i) PBPs bind analogs of the pheromone with some discrimination; (ii) PBPs experience enhancement of binding when presented with 1a or its enantiomer and 4a simultaneously; and (iii) the binding enhancement is also seen at high ligand:PBP ratios. We found no evidence of allostery, so the synergistic binding effects and the concentration effect may only be explained by multimerization of PBPs with each other, which leads to more than one population of binding sites. We suggest that the enhanced ligand binding at high ligand:PBP ratios may serve to sequester excess ligand and thereby attenuate very strong signals.     

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 PBPl-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.     

Pheromone-binding proteins contribute to the activation of olfactory receptor neurons in the silkmoths antheraea polyphemus and Bombyx mori

The sensilla trichodea of the silkmoth Antheraea polyphemus are innervated by three types of receptor neurons each responding specifically to one of three pheromone components. The sensillum lymph of these sensilla surrounding the sensory dendrites contains three different types of pheromone-binding proteins (PBPs) in high concentrations. The sensilla trichodea of the silkmoth Bombyx mori are supplied by two receptor neurons each tuned specifically to one of the two pheromone components bombykol and bombykal, but only one type of PBP has been found so far in these sensilla. Recombinant PBPs of both silkmoth species in various combinations with pheromone components were applied to the receptor neurons via tip-opened sensilla during electrophysiological recordings. Over a fairly broad range of pheromone concentrations the responses of the receptor neurons depended on both, the pheromone component and the type of the PBP. Therefore, the PBPs appear to contribute to the excitation of the receptor neurons. Furthermore, bombykal in combination with the expressed PBP of B. mori failed to activate the corresponding receptor neuron of B. mori, but did so if combined with one of the PBPs of A. polyphemus. Therefore, a still unknown binding protein involved in bombykal transport might be present in B. mori.     

Species-specific pheromonal compounds induce distinct conformational changes of pheromone binding protein subtypes from Antheraea polyphemus

We have investigated the structural features of three pheromone binding protein (PBP) subtypes from Antheraea polyphemus and monitored possible changes induced upon interaction with the Antheraea pheromonal compounds 4E,9Z-14:Ac [(E4,Z9)-tetradecadienyl-1-acetate], 6E,11Z-16:Ac [(E6,Z11)-hexadecadienyl-1-acetate], and 6E,11Z-16:Al [(E6,Z11)-hexadecadienal]. Circular dichroism and second derivative UV-difference spectroscopy data demonstrate that the structure of subtype PBP1 significantly changes upon binding of 4E,9Z-14:Ac. The related 6E,11Z-16:Ac was less effective and 6E,11Z-16:Al showed only a small effect. In contrast, in subtype PBP2 pronounced structural changes were only induced by the 6E,11Z-16:Al, and the subtype PBP3 did not show any considerable changes in response to the pheromonal compounds. The UV-spectroscopic data suggest that histidine residues are likely to be involved in the ligand-induced structural changes of the proteins, and this notion was confirmed by site-directed mutagenesis experiments. These results demonstrate that appropriate ligands induce structural changes in PBPs and provide evidence for ligand specificity of these proteins. Electronic Publication     

The solution NMR structure of Antheraea polyphemus PBP provides new insight into pheromone recognition by pheromone-binding proteins

Pheromone-binding proteins (PBPs) located in the antennae of male moth species play an important role in olfaction. They are carrier proteins, believed to transport volatile hydrophobic pheromone molecules across the aqueous sensillar lymph to the membrane-bound G protein-coupled olfactory receptor proteins. The roles of PBPs in molecular recognition and the mechanisms of pheromone binding and release are poorly understood. Here, we report the NMR structure of a PBP from the giant silk moth Antheraea polyphemus. This is the first structure of a PBP with specific acetate-binding function in vivo. The protein consists of nine alpha-helices: alpha1a (residues 2-5), alpha1b (8-12), alpha1c (16-23), alpha2 (27-34), alpha3a (46-52), alpha3b (54-59), alpha4 (70-79), alpha5 (84-100) and alpha6 (107-125), held together by three disulfide bridges: 19-54, 50-108 and 97-117. A large hydrophobic cavity is located inside the protein, lined with side-chains from all nine helices. The acetate-binding site is located at the narrow end of the cavity formed by the helices alpha3b and alpha4. The pheromone can enter this cavity through an opening between the helix alpha1a, the C-terminal end of the helix alpha6, and the loop between alpha2 and alpha3a. We suggest that Trp37 may play an important role in the initial interaction with the ligand. Our analysis also shows that Asn53 plays the key role in recognition of acetate pheromones specifically, while Phe12, Phe36, Trp37, Phe76, and Phe118 are responsible for non-specific binding, and Leu8 and Ser9 may play a role in ligand chain length recognition.     

中华蜜蜂性信息素结合蛋白ASP1的原核表达及配基结合特性分析

【目的】研究中华蜜蜂Apis cerana cerana信息素结合蛋白ASP1与蜜蜂信息素及某些植物挥发物分子的结合功能。【方法】构建中蜂ASP1的原核表达载体, 对其进行重组蛋白的诱导表达和分离纯化, 并得到具有生化活性的中蜂ASP1重组蛋白, 最后以1-NPN作为荧光报告探针, 通过荧光竞争结合实验研究中蜂重组ASP1蛋白与蜜蜂信息素及其他气味分子的结合功能。【结果】在22种潜在信息气味物质中, 有7种与中蜂ASP1有较强的结合能力, 能将1-NPN的相对荧光强度降至50%以下。其中发现蜂王信息素两种成分对 羟基苯甲酸甲酯和香草醇的竞争能力最强, 可分别引起1-NPN相对荧光值下降99.31%和95.50%, 解离常数K_D分别为13.39和98.44 μmol/L; 而与除蜂王信息素外的其他信息素如幼虫信息素和工蜂信息素等分子均不结合。此外中蜂ASP1对于水杨酸甲酯、 苯乙醛、 3, 4-二甲基苯甲醛4-烯丙基藜芦醚和β-紫罗兰酮等5种植物挥发物质能产生强度不一的结合。【结论】中蜂信息素结合蛋白ASP1对蜂王信息素具有非常强的特异性, 同时也能结合某些植物挥发性气味分子, 暗示中蜂ASP1是一种以蜂王信息素识别为主要功能、 植物挥发物识别为次要功能的多功能信息素结合蛋白。     

The pheromones of laying workers in two honeybee sister species: <Emphasis Type="Italic">Apis cerana</Emphasis> and <Emphasis Type="Italic">Apis mellifera</Emphasis>

When a honeybee colony loses its queen, workers activate their ovaries and begin to lay eggs. This is accompanied by a shift in their pheromonal bouquet, which becomes more queen like. Workers of the Asian hive bee Apis cerana show unusually high levels of ovary activation and this can be interpreted as evidence for a recent evolutionary arms race between queens and workers over worker reproduction in this species. To further explore this, we compared the rate of pheromonal bouquet change between two honeybee sister species of Apis cerana and Apis mellifera under queenright and queenless conditions. We show that in both species, the pheromonal components HOB, 9-ODA, HVA, 9-HDA, 10-HDAA and 10-HDA have significantly higher amounts in laying workers than in non-laying workers. In the queenright colonies of A. mellifera and A. cerana, the ratios (9-ODA)/(9-ODA + 9-HDA + 10-HDAA + 10-HDA) are not significantly different between the two species, but in queenless A. cerana colonies the ratio is significant higher than in A. mellifera, suggesting that in A. cerana, the workers’ pheromonal bouquet is dominated by the queen compound, 9-ODA. The amount of 9-ODA in laying A. cerana workers increased by over 585% compared with the non-laying workers, that is 6.75 times higher than in A. mellifera where laying workers only had 86% more 9-ODA compared with non-laying workers.     

Dose-dependent inhibition of emergency queen rearing by synthetic 9-ODA in the honey bee,Apis mellifera carnica

In the honey bee colony queen rearing is usually suppressed by releaser effects of the queen's pheromone. This is part of the dominance hierarchy maintaining the monogynous homeostasis. Under queenless conditions, the queen's control over the construction of emergency queen cells by the workers can be substitued by exposure to only one component of the mandibular pheromone secretion of a queen, the main compound (E)-9-oxo-2-decenoic acid. A novel and simple synthesis of (E)-9-oxo-2-decenoic acid is described, and a bioassay was developed by which a dose-dependent effect of synthetic (E)-9-oxo-2-decenoic acid presented on a dummy bee was evaluated.Abbreviation 9-ODA (E)-9-oxo-2-decenoic acid In memoriam Viktor Schwartz (1907–1992), Professor of Zoology and Developmental Biology, University of Tübingen, who introduced smoothened bee stings into microsurgery     

Reversing Evolution: Re-establishing Obligate Metal Ion Dependence in a Metal-independent KDO8P Synthase

Two distinct groups of 3-deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS), a key enzyme of cell-wall biosynthesis, differ by their requirement for a divalent metal ion for enzymatic activity. The unique difference between these groups is the replacement of the metal-binding Cys by Asn. Substitution of just this Asn for a Cys in metal-independent KDO8PS does not create the obligate metal-ion dependency of natural metal-dependent enzymes. We describe how three or four mutations of the metal-independent KDO8PS from Neisseria meningitidis produce a fully functional, obligately metal-dependent KDO8PS. For the substitutions Asn23Cys, Asp247Glu (this Asp binds to the metal ion in all metal-dependent KDO8PS) and Pro249Ala, and for double and triple combinations, mutant enzymes that contained Cys in place of Asn showed an increase in activity in the presence of divalent metal ions. However, combining these mutations with substitution by Ser of the Cys residue in the conserved ²⁴⁶CysAspGlyPro²⁴⁹ motif of metal-independent KDO8PS created enzymes with obligate metal dependency. The quadruple mutant (Asn23Cys/Cys246Ser/Asp247Glu/Pro249Ala) showed comparable activity to wild-type enzymes only in the presence of metal ions, with maximum activity with Cd²⁺, the metal ion that is strongly inhibitory at micromolar concentrations for the wild-type enzyme. In the absence of metal ions, activity was barely detectable for this quadruple mutant or for triple mutants bearing both Cys246Ser and Asn23Cys mutations. The structures of NmeKDO8PS and its Asn23Cys/Asp247Glu/Pro249Ala and quadruple mutants at pH 4.6 were characterized at resolutions better than 1.85 Å. Aged crystals of the Asn23Cys/Asp247Glu/Pro249Ala mutant featured a Cys23-Cys246 disulfide linkage, explaining the spectral bleaching observed when this mutant was incubated with Cu²⁺. Such bleaching was not observed for the quadruple mutant. Reverse evolution to a fully functional obligately metal-dependent KDO8PS has been achieved with just three directed mutations for enzymes that have, at best, 47% identity between metal-dependent and metal-independent pairs.     

How is Pheromone Specificity Encoded in Proteins?

Pheromone specificity in the Lepidoptera is encoded in proteincomponents of the antennal sensillum lymph and dendritic membrane.In this paper, we highlight recent work on the molecular determinantsof pheromone binding affinity of pheromone binding proteins(PBPS) of three genera. First, we describe new cDNA sequencesfor Lymantria dispar (Lymantriidae) and Agrotis segetum (Noctuidae).These data enrich the conclusions derived from our functionalstudies. Secondly, we indicate how preparation of multimilligramquantities of the recombinant PBP ‘Apol-3’ (originallyfrom Antheraea polyphemus) has provided a platform (i) to determinethe ligand binding sites using photoaffinity labeling, (ii)to conduct structural analysis by CD and NMR, and (iii) to measurebinding affinities using a new binding assay. Thirdly, we describethe use of expression-cassette PCR technology to prepare tworelated PBPS from Antheraea perneyi to test binding affinitiesof naturally-occurring homologous PBPs. Our results supporta model in which ligand specificity for chain length, doublebond position, and terminal functionality is partially encodedin the PBPS. We propose that the final decoding is accomplishedwhen the PBP-pheromone complex activates a G-protein coupledseven-transmembrane domain receptor that contains recognitionsites for both the presented pheromone and the presenting PBP.Chem. Senses 20: 461–469, 1995.     

Dissecting differential binding of fructose and phosphate as leaving group/nucleophile of glucosyl transfer catalyzed by sucrose phosphorylase

Site-directed mutagenesis was used to examine the specificity of Leuconostoc mesenteroides sucrose phosphorylase for utilization of fructose and phosphate as leaving group/nucleophile of the reaction. The largest catalytic defect in Arg(137)-->Ala (approximately 60-fold) and Tyr(340)-->Ala (approximately 2500-fold) concerned phosphate dependent half-reactions whereas that in Asp(338)-->Asn (approximately 7000-fold) derived from disruption of steps where fructose departs or attacks. The relative efficiencies for enzyme glucosylation by sucrose compared with alpha-d-glucose-1-phosphate and enzyme deglucosylation by phosphate compared with fructose were 5.5 and 6.2 for wild-type, 19 and 2.0 for Arg(137)-->Ala, 950 and 0.17 for Tyr(340)-->Ala, and 0.05 and 180 for Asp(338)-->Asn, respectively. Asp(338) and Tyr(340) have a key role in differential binding of fructose and phosphate, respectively.     

Discrimination of cis-trans sex pheromone components in two sympatric Lepidopteran species

Pheromone-binding proteins (PBPs) play an important role in the recognition of pheromones by insects. However, the abilities of these PBPs to discriminate pheromone components and recognize the isomers are unclear. Dendrolimus houi and Dendrolimus kikuchii are two sympatric coniferous pests whose pheromones have cis-trans isomers. We used these insect species to detect the precise recognition abilities of PBPs. The four PBPs examined showed male-biased antenna-intensive expression patterns, whereas PBP1 showed higher expression than PBP2 in the antenna. DhouPBP1 only bound to a minor interspecific pheromone component, whereas DhouPBP2 bound to all three intraspecific components and another minor interspecific component. DkikPBP1 and DkikPBP2 could recognize all three intraspecific components with affinities negatively correlated with their ratios, and they bound to interspecific pheromones with affinity that was positively correlated with the ratios. The four PBPs have different cis-trans isomer discrimination abilities, i.e., DhouPBP1 and DkikPBP1 could not discriminate the two cis-trans isomer pairs of pheromones from the two species, whereas DhouPBP2 could discriminate between both pairs, and DkikPBP2 could only discriminate one pair. Overall, PBPs from D. houi and D. kikuchii use different strategies to help the moths to discriminate the intra- and interspecific pheromone components. Our work will contribute to better understanding of the sex pheromone recognition mechanism in these two sister species of moths and provide insights into more effective management practices of these pest species.