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The Microbiome Protocols eBook (MPB) serves as a crucial bridge, filling gaps in microbiome protocols for both wet experiments and data analysis. The first edition, launched in 2020, featured 152 meticulously curated protocols, garnering widespread acclaim. We now extend a sincere invitation to researchers to participate in the upcoming 2nd version of MPB, contributing their valuable protocols to advance microbiome research.
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In this work, we introduced a siderophore information database (SIDERTE), a digitized siderophore information database containing 649 unique structures. Leveraging this digitalized data set, we gained a systematic overview of siderophores by their clustering patterns in the chemical space. Building upon this, we developed a functional group-based method for predicting new iron-binding molecules with experimental validation. Expanding our approach to the collection of open natural products (COCONUT) database, we predicted a staggering 3199 siderophore candidates, showcasing remarkable structure diversity that is largely unexplored. Our study provides a valuable resource for accelerating the discovery of novel iron-binding molecules and advancing our understanding of siderophores.
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Expressions of a wide range of cytoprotective counter-defense genes are mainly regulated by the Keap1-Nrf2-ARE signaling pathway in response to oxidative stress from xenobiotics. Gossypol is the major antiherbivore secondary metabolite of cotton, but how the polyphagous pest Helicoverpa armigera copes with this phytochemical to utilize its favorite host plant cotton remains largely elusive. In this study, we first suppressed the Keap1 gene in newly hatched larvae of cotton bollworm by feeding them the siRNA diet for 4 days. All of the larvae were subsequently fed the artificial diet supplied with gossypol or the control diet for 5 days. We identified that the knockdown of the Keap1 gene significantly decreased larval mortality and significantly increased the percentages of larval survival, reaching the fourth instar, compared with ncsiRNA when exposed to a diet containing gossypol. Three counter-defense genes CYP9A17, CYP4L11 and UGT41B3, which were related to the induction or metabolism of gossypol according to the report before, were all significantly up-regulated after the knockdown of the Keap1 gene. The Antioxidant Response Elements (AREs) were also detected in the promoter regions of the three counter-defense genes above. These data indicate that the suppression of the Keap1 gene activates the Keap1-Nrf2-ARE signaling pathway, up-regulates the expressions of counter-defense genes involved in the resistance of oxidative stress and finally contributes to reducing the susceptibility of gossypol. Our results provide more knowledge about the transcriptional regulation mechanisms of counter-defense genes that enable the cotton bollworm to adapt to the diversity of host plants including cotton.
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Moth insects rely on sex pheromones for long distance attraction and searching for sex partners. The biosynthesis of moth sex pheromones involves the catalytic action of multiple enzymes, with desaturases playing a crucial role in the process of carbon chain desaturation. However, the specific desaturases involved in sex pheromone biosynthesis in fall armyworm (FAW), Spodoptera frugiperda, have not been clarified. In this study, a Δ11 desaturase (SfruDES1) gene in FAW was knocked out using the CRISPR/Cas9 genome editing system. A homozygous mutant of SfruDES1 was obtained through genetic crosses. The gas chromatography-mass spectrometry (GC-MS) analysis results showed that the three main sex pheromone components (Z7-12:Ac, Z9-14:Ac, and Z11-16:Ac) and the three minor components (Z9-14:Ald, E11-14:Ac and Z11-14:Ac) of FAW were not detected in homozygous mutant females compared to the wild type. Furthermore, behavioral assay demonstrated that the loss of SfruDES1 resulted in a significant reduction in the attractiveness of females to males, along with disruptions in mating behavior and oviposition. Additionally, in a heterologous expression system, recombinant SfruDES1 could introduce a cis double bond at the Δ11 position in palmitic acid, which resulted in the changes in components of the synthesized products. These findings suggest desaturase plays a key role in the biosynthesis of sex pheromones, and knockout of the SfruDES1 disrupts sex pheromone biosynthesis and mating behavior in FAW. The SfruDES1 could serve as tool to develop a control method for S. frugiperda.
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Mariposas , Atrativos Sexuais , Animais , Feminino , Masculino , Spodoptera/genética , Spodoptera/metabolismo , Atrativos Sexuais/metabolismo , Oviposição , Mariposas/genética , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/metabolismoRESUMO
Recent studies revealed that insect chemosensory proteins (CSPs) both play essential roles in insect olfaction and insect resistance. However, functional evidence supporting the crosslink between CSP and insecticide resistance remains unexplored. In the present study, 22 SfruCSP transcripts were identified from the fall armyworm (FAW) and SfruCSP1 and SfruCSP2 are enriched in the larval cuticle and could be induced by multiple insecticides. Both SfruCSP1 and SfruCSP2 are highly expressed in the larval inner endocuticle and outer epicuticle, and these two proteins exhibited high binding affinities with three insecticides (chlorfenapyr, chlorpyrifos and indoxacarb). The knockdown of SfruCSP1 and SfruCSP2 increased the susceptibility of FAW larvae to the above three insecticides, and significantly increased the penetration ratios of these insecticides. Our in vitro and in vivo evidence suggests that SfruCSP1 and SfruCSP2 are insecticide binding proteins and confer FAW larval resistance to chlorfenapyr, chlorpyrifos and indoxacarb by an insecticide sequestration mechanism. The study should aid in the exploration of larval cuticle-enriched CSPs for insect resistance management.
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Proteínas de Insetos , Resistência a Inseticidas , Inseticidas , Larva , Oxazinas , Spodoptera , Animais , Spodoptera/efeitos dos fármacos , Spodoptera/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Clorpirifos/farmacologiaRESUMO
Microbes shape their habitats by consuming resources and producing a diverse array of chemicals that can serve as public goods. Despite the risk of exploitation by cheaters, genes encoding sharable molecules like siderophores are widely found in nature, prompting investigations into the mechanisms that allow producers to resist invasion by cheaters. In this work, we presented the chemostat-typed "resource partition model" to demonstrate that dividing the iron resource between private and public siderophores can promote stable or dynamic coexistence between producers and cheaters in a well-mixed environment. Moreover, our analysis shows that when microbes not only consume but also produce resources, chemical innovation leads to stability criteria that differ from those of classical consumer resource models, resulting in more complex dynamics. Our work sheds light on the role of chemical innovations in microbial communities and the potential for resource partition to facilitate dynamical coexistence between cooperative and cheating organisms.
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Olfaction plays a crucial role in locating food sources, mates, and spawning sites in the fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae). In the current study, SfruOR14, a highly conserved odorant receptor (OR) in lepidopteran species, was newly uncovered in S. frugiperda. In two-electrode voltage clamp recordings, the SfruOR14/Orco complex was narrowly tuned to six volatile compounds including phenylacetaldehyde (PAA), benzaldehyde, heptaldehyde, (E)-2-hexen-1-al, cinnamaldehyde, and 2-phenylethanol, among which PAA showed the strongest binding affinity. Subsequent homology modeling and molecular docking revealed that Phe79, His83, Tyr149, Pro176, Gln177, Leu202, and Thr348 in SfruOR14 were the key binding residues against the six ligands. Finally, as a result of site-directed mutagenesis, the SfruOR14His83Ala mutant completely lost its binding capabilities toward all ligands. Taken together, our findings provide valuable insights into understanding the interaction between SfruOR14 and the chemical ligands including PAA, which can help to design novel olfactory modulators for pest control.
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Mutação Puntual , Receptores Odorantes , Animais , Spodoptera , Ligantes , Simulação de Acoplamento MolecularRESUMO
Scleroderma guani is a generalist ectoparasitoid of wood-boring insects. The chemosensory genes expressed in its antennae play crucial roles in host-seeking. In the present study, we identified 14 OBP genes for the first time from the antennae transcriptomes and genomic data of S. guani. The expression profiles of 14 OBPs were tested by RT-qPCR, and the RT-qPCR results showed that SguaOBP2/5/6/11/12/13 were specifically highly expressed in the female antennae. Then we performed ligand binding assays to test the interactions between six selected SguaOBPs with host specific chemical compounds from M. alternatus and pines. The binding results indicated that SguaOBP12 had a higher binding affinity with longifolene, ß-caryophyllene, α-pinene, ß-pinene, myrcene, butylated hydroxytoluene, and 3-carene. SguaOBP11 had a high or medium binding affinity with them. Furthermore, both SguaOBP11 and SguaOBP12 had a medium binding affinity with the aggregation pheromone of Monochamus species, 2-undecyloxy-1-ethanol. Finally, by using molecular docking and RNAi, we further explored the molecular interactions and behavioral functions of SguaOBP11 and SguaOBP12 with these vital odor molecules. Our study contributes to the further understanding of chemical communications between S. guani and its host, and further exploration for its role as a more effective biological control agent.
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Besouros , Receptores Odorantes , Vespas , Feminino , Animais , Vespas/genética , Odorantes , Simulação de Acoplamento Molecular , Besouros/genética , Feromônios , Proteínas de Insetos/metabolismo , Receptores Odorantes/química , FilogeniaRESUMO
Non-ribosomal peptide synthetase (NRPS) is a diverse family of biosynthetic enzymes for the assembly of bioactive peptides. Despite advances in microbial sequencing, the lack of a consistent standard for annotating NRPS domains and modules has made data-driven discoveries challenging. To address this, we introduced a standardized architecture for NRPS, by using known conserved motifs to partition typical domains. This motif-and-intermotif standardization allowed for systematic evaluations of sequence properties from a large number of NRPS pathways, resulting in the most comprehensive cross-kingdom C domain subtype classifications to date, as well as the discovery and experimental validation of novel conserved motifs with functional significance. Furthermore, our coevolution analysis revealed important barriers associated with re-engineering NRPSs and uncovered the entanglement between phylogeny and substrate specificity in NRPS sequences. Our findings provide a comprehensive and statistically insightful analysis of NRPS sequences, opening avenues for future data-driven discoveries.
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Peptídeo Sintases , Peptídeos , Peptídeos/química , Peptídeo Sintases/genética , Peptídeo Sintases/química , Peptídeo Sintases/metabolismoRESUMO
Bacteria secrete siderophores whose function is to acquire iron. In recent years, the siderophores of several Chryseobacterium species were shown to promote the health and growth of various plants such as tomato or rice. However, the chemical nature of Chryseobacterium siderophores remained unexplored despite great interest. In this work, we present the purification and structure elucidation by nuclear magnetic resonance (NMR) spectroscopy and tandem mass spectrometry (MS/MS) of chryseochelin A, a novel citrate-based siderophore secreted by three Chryseobacterium strains involved in plant protection. It contains the unusual building blocks 3-hydroxycadaverine and fumaric acid. Furthermore, the unstable structural isomer chryseochelin B and its stable derivative containing fatty acid chains, named chryseochelin C, were identified by mass spectrometric methods. The latter two incorporate an unusual ester connectivity to the citrate moiety showing similarities to achromobactin from the plant pathogen Dickeya dadantii. Finally, we show that chryseochelin A acts in a concentration-dependent manner against the plant-pathogenic Ralstonia solanacearum strain by reducing its access to iron. Thus, our study provides valuable knowledge about the siderophores of Chryseobacterium strains, which have great potential in various applications.
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Chryseobacterium , Sideróforos , Sideróforos/química , Ácido Cítrico , Espectrometria de Massas em Tandem , Ferro , CitratosRESUMO
The green bug Apolygus lucorum is a notorious pest that feeds on multiple crops, including fruit trees, vegetables, and cotton. The odorant-binding proteins (OBPs) are considered to perform crucial roles in regulating A. lucorum behaviors such as mating and feeding. In this study, we first identified OBPs in the A. lucorum genome. Then, we calculated the expression levels of these OBP genes in different tissues and stages. Thereafter, we conducted ligand-binding assay to test the interactions between nine selected AlucOBPs and multiple chemical compounds. The result showed that there were 31 OBP genes encoding 39 transcripts in the A. lucorum genome, and several OBP clusters were found. Comprehensive expression profiling revealed the tissue-specific expression of some OBP genes. The results of fluorescence competitive binding assays showed that these nine AlucOBPs could specifically bind to plant volatiles, nonvolatile compounds, and synthetic analogs thereof. Additionally, AlucOBP19 was suggested to function in gustatory sensing to avoid deleterious plant secondary metabolites, as AlucOBP19 showed high expression in the mouthparts and legs and could interact with quercetin. Our findings highlight the potential biotechnological application of plant volatiles and their synthetic analogs as ecological attractants and provide new gene targets for control of A. lucorum.
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Heterópteros , Receptores Odorantes , Animais , Odorantes , Heterópteros/genética , Receptores Odorantes/genética , Comunicação Celular , Verduras , Produtos Agrícolas/metabolismo , Proteínas de Insetos/genéticaRESUMO
Recent studies have indicated that the plant volatile methyl benzoate (MB) exhibits significant insecticidal bioactivity against several common insects. However, the potential environmental hazards of MB and its safety to non-target organisms is poorly understood. In the present study, these characteristics were investigated through laboratory experiments and field investigations. The results revealed that MB was highly toxic to the agricultural pest, fall armyworm Spodoptera frugiperda. Compared with the commercial pesticide lambda-cyhalothrin, the toxicities of MB against S. frugiperda larvae and adults were comparable and 3.41 times higher, respectively. Behavioral bioassays showed that the percentage repellency of MB to S. frugiperda larvae was 56.72 %, and MB induced 69.40 % oviposition deterrence rate in S. frugiperda female adults. Furthermore, in terms of median lethal concentration (LC50) and median lethal doses (LD50), MB exhibited non-toxic effects on non-target animals with 3-d LC50 of > 1 % to natural predators (Coccinella septempunctata and Harmonia axyridis), 3-d LD50 of 467.86 µg/bee to the bumblebee Bombus terrestris, 14-d LC50 of 971.09 mg/kg to the earthworm Eisenia fetida, and 4-d LC50 of 47.30 mg/L to the zebrafish Brachydanio rerio. The accumulation of MB in the soil and earthworms was found to be extremely limited. Our comparative study clearly demonstrated that MB is effective as a selective botanical pesticide against S. frugiperda and it is safe to use in the tested environment, with no toxic effects on non-target animals and natural predators.
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Besouros , Inseticidas , Oligoquetos , Animais , Benzoatos , Feminino , Inseticidas/toxicidade , Larva , Solo , Spodoptera , Peixe-ZebraRESUMO
Persistent microbial symbioses can confer greater fitness to their host under unfavorable conditions, but manipulating such beneficial interactions necessitates a mechanistic understanding of the consistently important microbiomes for the plant. Here, we examined the phylogenetic profiles and plant-beneficial traits of the core microbiota that consistently inhabits the rhizosphere of four divergent Cd hyperaccumulators and an accumulator. We evidenced the existence of a conserved core rhizosphere microbiota in each plant distinct from that in the non-hyperaccumulating plant. Members of Burkholderiaceae and Sphingomonas were the shared cores across hyperaccumulators and accumulators. Several keystone taxa in the rhizosphere networks were part of the core microbiota, the abundance of which was an important predictor of plant Cd accumulation. Furthermore, an inoculation experiment with synthetic communities comprising isolates belonging to the shared cores indicated that core microorganisms could facilitate plant growth and metal tolerance. Using RNA-based stable isotope probing, we discovered that abundant core taxa overlapped with active rhizobacteria utilizing root exudates, implying that the core rhizosphere microbiota assimilating plant-derived carbon may provide benefits to plant growth and host phenotype such as Cd accumulation. Our study suggests common principles underpinning hyperaccumulator-microbiome interactions, where plants consistently interact with a core set of microbes contributing to host fitness and plant performance. These findings lay the foundation for harnessing the persistent root microbiomes to accelerate the restoration of metal-disturbed soils.
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Metais Pesados , Microbiota , Bactérias/genética , Cádmio , Carbono , Filogenia , Raízes de Plantas/microbiologia , Plantas/genética , RNA , Rizosfera , Solo , Microbiologia do SoloRESUMO
The green peach aphid Myzus persicae (Hemiptera: Aphididae) relies heavily on its olfactory system to locate plant hosts, find mates, and avoid parasitoids or predators. The insect odorant receptors (ORs) have been proven to play a critical role in the perception of odorants from the environment. In the present study, 33 odorant receptor candidate genes including the Orco gene were identified from the antennal, head, legs and body transcriptomes of M. persicae. Phylogenetic analysis of ORs from seven different orders of insect species suggests that ORs from different insect species are highly divergent and most ORs from the same species formed monophyletic groups. In addition, the aphid ORs were clustered into six different sub-clades in the same clade. Furthermore, the genomic structure of the OR genes also tends to be consistent, suggesting that ORs from the family Aphididae have a relatively close evolutionary relationship. Reads per kilobase per million (RPKM) and tissue expression profiles analyses revealed that 27 out of the 33 MperORs were uniquely or primarily expressed in the antennae, indicating their putative roles in chemoreception. This work provides a foundation to further investigate the molecular and ecological functions of MperORs in the aphid-aphid, aphid-plant and aphid-natural enemy interactions.
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The rhizosphere microbiome forms a first line of defense against soilborne pathogens. To date, most microbiome enhancement strategies have relied on bioaugmentation with antagonistic microorganisms that directly inhibit pathogens. Previous studies have shown that some root-associated bacteria are able to facilitate pathogen growth. We therefore hypothesized that inhibiting such pathogen helpers may help reduce pathogen densities. We examined tripartite interactions between a model pathogen, Ralstonia solanacearum, two model helper strains and a collection of 46 bacterial isolates recovered from the tomato rhizosphere. This system allowed us to examine the importance of direct (effects of rhizobacteria on pathogen growth) and indirect (effects of rhizobacteria on helper growth) pathways affecting pathogen growth. We found that the interaction between rhizosphere isolates and the helper strains was the major determinant of pathogen suppression both in vitro and in vivo. We therefore propose that controlling microbiome composition to prevent the growth of pathogen helpers may become part of sustainable strategies for pathogen control.
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Microbiota , Ralstonia solanacearum , Solanum lycopersicum , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Ralstonia solanacearum/fisiologia , RizosferaRESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme involved in the ubiquitous process of glycolysis and presents a loop (residues 208-215 of Escherichia coli GAPDH) in two alternative conformations (I and II). It is uncertain what triggers this loop rearrangement, as well as which is the precise site from which phosphate attacks the thioacyl intermediate precursor of 1,3-bisphosphoglycerate (BPG). To clarify these uncertainties, we determined the crystal structures of complexes of wild-type GAPDH (WT) with NAD and phosphate or G3P, and of essentially inactive GAPDH mutants (C150S, H177A), trapping crystal structures for the thioacyl intermediate or for ternary complexes with NAD and either phosphate, BPG, or G3P. Analysis of these structures reported here lead us to propose that phosphate is located in the "new Pi site" attacks the thioester bond of the thioacyl intermediate to generate 1,3-bisphosphoglyceric acid (BPG). In the structure of the thioacyl intermediate, the mobile loop is in conformation II in subunits O, P, and R, while both conformations coexist in subunit Q. Moreover, only the Q subunit hosts bound NADH. In the R subunit, only the pyrophosphate part of NADH is well defined, and NADH is totally absent from the O and P subunits. Thus, the change in loop conformation appears to occur after NADH is produced, before NADH is released. In addition, two new D-glyceraldehyde-3-phosphate (G3P) binding forms are observed in WT.NAD.G3P and C150A+H177A.NAD.G3P. In summary, this paper improves our understanding of the GAPDH catalytic mechanism, particularly regarding BPG formation.
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Escherichia coli , Gliceraldeído-3-Fosfato Desidrogenases , NADRESUMO
Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds of cellobiose, resulting in the production of glucose, which is an important step for the effective utilization of cellulose. In the present study, a thermostable ß-glucosidase was isolated and purified from the Thermoprotei Thermofilum sp. ex4484_79 and subjected to enzymatic and structural characterization. The purified ß-glucosidase (TsBGL) exhibited maximum activity at 90°C and pH 5.0 and displayed maximum specific activity of 139.2µmol/min/mgzne against p-nitrophenyl ß-D-glucopyranoside (pNPGlc) and 24.3µmol/min/mgzen against cellobiose. Furthermore, TsBGL exhibited a relatively high thermostability, retaining 84 and 47% of its activity after incubation at 85°C for 1.5h and 90°C for 1.5h, respectively. The crystal structure of TsBGL was resolved at a resolution of 2.14Å, which revealed a classical (α/ß)8-barrel catalytic domain. A structural comparison of TsBGL with other homologous proteins revealed that its catalytic sites included Glu210 and Glu414. We provide the molecular structure of TsBGL and the possibility of improving its characteristics for potential applications in industries.
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We retrospectively analyzed the clinical data of 635 patients with acute acromioclavicular dislocation, who underwent surgery in our hospital between May 2014 and June 2020. Patients were divided into group A (clavicular hook plate) and group B (Triple-Endobutton plates via double-incision). The propensity score analysis using one to one match was performed for comparisons. We obtained 292 matched patients' data. The matched preoperative clinical characteristics were a balance between the two groups. All clinical parameters showed insignificant differences (P > 0.05). Compared with group A, group B has longer operative time (P < 0.001) and more blood loss (P < 0.001); however, the mean incision length (P < 0.001) and length of hospitalization (P < 0.001) were shorter in group B than in the group A. The mean VAS in group B were significantly lower than in group A at each time point (P < 0.001), and the UCLA shoulder score was higher in the group B. The CMS scores were also higher in group B than in group A, including before removal and 12 weeks after removal (P < 0.001). The clinical efficacy of the double-incision Triple-Endobutton plate is better than the clavicular hook plate technology, and achieves anatomical reduction by reconstructing coracoclavicular ligament.
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In order to study the performance of the bamboo fiber composites prepared by filament winding, composites reinforced with jute fiber and glass fiber were used as control samples. The structure and mechanical properties of the composites were investigated by scanning electric microscope (SEM), tensile testing, bending testing, and dynamic mechanical analysis. The results demonstrated that the bamboo fiber composites exhibited lower density (0.974 g/cm3) and mechanical properties in comparison of to fiber composite and glass fiber composite, because the inner tissue structure of bamboo fiber was preserved without resin adsorbed into the cell cavity of fibrous parenchyma. The bamboo fibers in composites were pulled out, while the fibers in the surface of composites were torn, resulting in the lowest mechanical performance of bamboo fiber composites. The glass transition temperature of twisting bamboo fiber Naval Ordnance Laboratory (TBF-NOL) composite (165.89 °C) was the highest in general, which indicated that the TBF circumferential composite had the best plasticizing properties and better elasticity, the reason being that the fiber-reinforced epoxy circumferential composite interface joint is a physical connection, which restricts the movement of the molecular chain of the epoxy matrix, making the composite have a higher storage modulus (6000 MPa). In addition, The TBF-NOL had the least frequency dependence, and the circumferential composite prepared by TBF had the least performance variability. Therefore, the surface and internal structures of the bamboo fiber should be further processed and improved by decreasing the twisting bamboo fiber (TBF) diameter and increasing the specific surface area of the TBF and joint surface between fibers and resin, to improve the comprehensive properties of bamboo fiber composites.
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Sex pheromones are deemed to play a significant role in sexual communication of most insects. Although many sex pheromone components in mirid bugs have been identified, the roles of odorant receptors in sex pheromone perception in Adelphocoris spp. (Hemiptera: Miridae) remain unknown so far. Here, AlinOR33, a candidate sex pheromone receptor in Adelphocoris lineolatus was functionally characterized. Phylogenetic analysis showed that AlinOR33 clustered with the sex pheromone receptor AlucOR4 fromApolygus lucorum. Quantitative real-time PCR measurement revealed that the expression of AlinOR33 increased gradually from nymph to adult stage and reached its peak in the antennae of 3-day-old mated male bugs. The subsequent in situ hybridization demonstrated that AlinOR33 was mainly expressed in sensilla trichoid on the antennae of A. lineolatus. In the two-electrode voltage clamp recordings, AlinOR33/AlinOrco was specifically tuned to four sex pheromone components including butyl butyrate, hexyl hexanoate, trans-2-hexenyl butyrate and hexyl butyrate, and especially most sensitive to the major component trans-2-hexenyl butyrate. After dsAlinOR33 injection, the electroantennogram responses of males to four sex pheromone components were reduced significantly (â¼50%). Compared to control bugs, dsAlinOR33-injected male bugs almost lost behavioral preference for trans-2-hexenyl butyrate. Furthermore, the wingbeat frequency of dsAlinOR33-injected male bugs notably declined. Therefore, we conclude that as a candidate sex pheromone receptor, AlinOR33 plays essential roles in the sexual behavior of A. lineolatus.