Plant volatile compound methyl benzoate is highly effective against Spodoptera frugiperda and safe to non-target organisms as an eco-friendly botanical-insecticide.

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.

The terpene synthase gene family in Gossypium hirsutum harbors a linalool synthase GhTPS12 implicated in direct defence responses against herbivores.

Herbivore-induced terpenes have been reported to function as ecological signals in plant-insect interactions. Here, we showed that insect-induced cotton volatile blends contained 16 terpenoid compounds with a relatively high level of linalool. The high diversity of terpene production is derived from a large terpene synthase (TPS) gene family. The TPS gene family of Gossypium hirsutum and Gossypium raimondii consist of 46 and 41 members, respectively. Twelve TPS genes (GhTPS4-15) could be isolated, and protein expression in Escherichia coli revealed catalytic activity for eight GhTPS. The upregulation of the majority of these eight genes additionally supports the function of these genes in herbivore-induced volatile biosynthesis. Furthermore, transgenic Nicotiana tabacum plants overexpressing GhTPS12 were generated, which produced relatively large amounts of (3S)-linalool. In choice tests, female adults of Helicoverpa armigera laid fewer eggs on transgenic plants compared with non-transformed controls. Meanwhile, Myzus persicae preferred feeding on wild-type leaves over leaves of transgenic plants. Our findings demonstrate that transcript accumulation of multiple TPS genes is mainly responsible for the production and diversity of herbivore-induced volatile terpenes in cotton. Also, these genes might play roles in plant defence, in particular, direct defence responses against herbivores.

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.

Aminopeptidase N5 (APN5) as a Putative Functional Receptor of Cry1Ac Toxin in the Larvae of Athetis lepigone.

Athetis lepigone was a new lepidopteran pest and caused severe damage to maize crops in China. We have detected that Cry1Ac protoxin and toxin were highly active against the larvae of A. lepigone. However, there is no report about the mode of action of Bt Cry1Ac toxin against this pest until now. A 110 kDa APN5 protein from BBMV of A. lepigone was identified as the binding receptor of Cry1Ac toxin using Ligand blotting. The Cry1Ac receptor APN5 was cloned from A. lepigone larval midgut mRNA and named as AlAPN5 (GenBank accession no.: KU950745). AlAPN5 had a GATEN motif and been classified to Class 5 APNs. 79.2% reduction in mortality was observed when A. lepigone larvae were injected with siRNA of the AlAPN5 gene and treated with Cry1Ac toxin. These data demonstrate that AlAPN5 is a putative functional receptor and maybe the only receptor of Cry1Ac in A. lepigone.

Identification of genes expressed in the sex pheromone gland of the black cutworm Agrotis ipsilon with putative roles in sex pheromone biosynthesis and transport.

BACKGROUND: One of the challenges in insect chemical ecology is to understand how insect pheromones are synthesised, detected and degraded. Genome wide survey by comparative sequencing and gene specific expression profiling provide rich resources for this challenge. A. ipsilon is a destructive pest of many crops and further characterization of the genes involved in pheromone biosynthesis and transport could offer potential targets for disruption of their chemical communication and for crop protection. RESULTS: Here we report 454 next-generation sequencing of the A. ipsilon pheromone gland transcriptome, identification and expression profiling of genes putatively involved in pheromone production, transport and degradation. A total of 23473 unigenes were obtained from the transcriptome analysis, 86% of which were A. ipsilon specific. 42 transcripts encoded enzymes putatively involved in pheromone biosynthesis, of which 15 were specifically, or mainly, expressed in the pheromone glands at 5 to 120-fold higher levels than in the body. Two transcripts encoding for a fatty acid synthase and a desaturase were highly abundant in the transcriptome and expressed more than 40-fold higher in the glands than in the body. The transcripts encoding for 2 acetyl-CoA carboxylases, 1 fatty acid synthase, 2 desaturases, 3 acyl-CoA reductases, 2 alcohol oxidases, 2 aldehyde reductases and 3 acetyltransferases were expressed at a significantly higher level in the pheromone glands than in the body. 17 esterase transcripts were not gland-specific and 7 of these were expressed highly in the antennae. Seven transcripts encoding odorant binding proteins (OBPs) and 8 encoding chemosensory proteins (CSPs) were identified. Two CSP transcripts (AipsCSP2, AipsCSP8) were highly abundant in the pheromone gland transcriptome and this was confirmed by qRT-PCR. One OBP (AipsOBP6) were pheromone gland-enriched and three OBPs (AipsOBP1, AipsOBP2 and AipsOBP4) were antennal-enriched. Based on these studies we proposed possible A. ipsilon biosynthesis pathways for major and minor sex pheromone components. CONCLUSIONS: Our study identified genes potentially involved in sex pheromone biosynthesis and transport in A. ipsilon. The identified genes are likely to play essential roles in sex pheromone production, transport and degradation and could serve as targets to interfere with pheromone release. The identification of highly expressed CSPs and OBPs in the pheromone gland suggests that they may play a role in the binding, transport and release of sex pheromones during sex pheromone production in A. ipsilon and other Lepidoptera insects.

The preferential binding of a sensory organ specific odorant binding protein of the alfalfa plant bug Adelphocoris lineolatus AlinOBP10 to biologically active host plant volatiles.

Semiochemicals such as sex pheromones and plant volatiles are crucial components of insect mating systems and host plant localization. In the olfactory signal transduction pathway, odorant-binding proteins (OBPs) are important elements that function in the first step of the pathway by carrying hydrophobic semiochemicals across the sensillum lymph to the olfactory receptors (ORs). In this study, we examined the binding affinities of semiochemicals to AlinOBP10, a putative OBP from the alfalfa plant bug, Adelphocoris lineolatus, that we demonstrate is expressed mainly in sensory organs. We then characterized the biological activities of the high affinity semiochemicals by measuring their electrophysiological activities in antennae and behavioral responses in the plant bug. AlinOBP10 displayed weak binding affinities to two major putative pheromone components, hexyl butyrate and (E)-2-hexenyl butyrate. In contrast, AlinOBP10 exhibited higher binding affinities to six host plant volatiles, namely myrcene, ß-pinene, ß-ionone, 3-hexanone, (E)-2-hexenal, and 1-hexanol. The biological activities of these six putative ligands were further studied in electroantennogram recordings and Y-tube olfactometer trials. The three compounds, (E)-2-hexenal, 1-hexanol, and 3-hexanone elicited strong electrophysiological responses, but elicited distinct behaviors. While 3-hexanone was attractive to female adults, (E)-2-hexenal and 1-hexanol were significant repellents. Although a weak electrophysiological response was elicited with ß-pinene, it was a strong repellent. These results demonstrate that AlinOBP10 can interact with attractants, as well as repellents, with some specificity toward plant volatiles over sex pheromones.

Mapping of minimal motifs of B-cell epitopes on human zona pellucida glycoprotein-3.

The human zona pellucida glycoprotein-3 (hZP3) by virtue of its critical role during fertilization has been proposed as a promising candidate antigen to develop a contraceptive vaccine. In this direction, it is imperative to map minimal motifs of the B cell epitopes (BCEs) so as to avoid ZP-specific oophoritogenic T cell epitopes (TCEs) in the ZP3-based immunogens. In this study, based on known results of mapping marmoset and bonnet monkey ZP3 (mstZP3 and bmZP3), two predictable epitopes(23-30 and 301-320) on hZP3 were first confirmed and five minimal motifs within four epitopes on hZP3 were defined using serum to recombinant hZP3a(22-176) or hZP3b(177-348) as well as a biosynthetic peptide strategy. These defined minimal motifs were QPLWLL(23-28) for hZP3(23-30), MQVTDD(103-108) for hZP3(93-110), EENW(178-181) for hZP3(172-190), as well as SNSWF(306-310) and EGP(313-315) for hZP3(301-320), respectively. Furthermore, the antigenicity of two peptides for hZP3(172-187) and hZP3(301-315) and specificity of the antibody response to these peptides were also evaluated, which produced high-titer antibodies in immunized animals that were capable of reacting to ZP on human oocytes, r-hZP3b(177-348) protein, as well as r-hZP3(172-190), r-hZP3(303-310), and r-hZP3(313-320) epitope peptides fused with truncated GST188 protein.

Binding characterization of recombinant odorant-binding proteins from the parasitic wasp, Microplitis mediator (Hymenoptera: Braconidae).

Chemoreception in insects is mediated by small odorant-binding proteins (OBPs) that are believed to carry lipophilic stimuli to the olfactory receptor cells through the aqueous sensillar lymph. Binding experiments and recent structural studies of OBPs have illustrated their versatility and ability to accommodate ligands of different shapes and chemical structures. We expressed and purified seven recombinant OBPs (MmedOBP1-MmedOBP7) from the parasitic wasp, Microplitis mediator (Hymenoptera: Braconidae) in a prokaryotic expression system. With 4,4'-dianilino-1,1'-binaphthyl-5,5'-sulfonic acid (bis-ANS) as a fluorescent probe, the ligand-binding specificities of these seven MmedOBPs with 50 small organic compounds were investigated in vitro. The results revealed that all of the M. mediator OBPs can bind a wide variety of odorant molecules with different binding affinities. The best ligand for all seven MmedOBPs was ß-ionone. MmedOBP2 showed affinity for some aromatic compounds, whereas MmedOBP4 and MmedOBP6 bound several terpenoids. MmedOBP5 bound ß-ionone, but did not bind any of the other potential ligands that we tested.

Functional characterization and immunolocalization of odorant binding protein 1 in the lucerne plant bug, Adelphocoris lineolatus (GOEZE).

In the insect phylum, the relationships between individuals and their environment are often modulated by chemical communication. Odorant binding proteins (OBPs) are widely and robustly expressed in insect olfactory organs and play a key role in chemosensing and transporting hydrophobic odorants across the sensillum lymph to the olfactory receptor neuron. In this study, a novel OBP gene (AlinOBP1) in the lucerne plant bug, Adelphocoris lineolatus was identified, cloned and expressed. Real-time PCR results indicated that the expression level of AlinOBP1 gene differed in each developmental stage (from first instar to adult) and was predominantly expressed in the antennae of adults. The expression level of AlinOBP1 was 1.91 times higher in male antennae than in female antennae. The binding properties of AlinOBP1 with 114 odorants were measured using a fluorescence probe, N-phenyl-1-naphthylamine (1-NPN), with fluorescence competitive binding. The results revealed that AlinOBP1 exhibits high binding abilities with two major putative pheromone components, ethyl butyrate and trans-2-hexenyl butyrate. In addition, it was observed that six volatiles released from cotton, octanal, nonanal, decanal, 2-ethyl-1-hexanol, ß-caryophyllene and ß-ionone also bind to AlinOBP1. Immunocytochemistry analysis showed that AlinOBP1 was expressed in the sensillum lymph of sensilla trichodica and sensilla basiconca. Our results demonstrate that AlinOBP1 may function as a carrier in the chemoperception of the lucerne plant bug.

Functional Characterization of a Candidate Sex Pheromone Receptor AlinOR33 Involved in the Chemoreception of Adelphocoris lineolatus.

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 (∼50%). 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.

Coordinative mediation of the response to alarm pheromones by three odorant binding proteins in the green peach aphid Myzus persicae.

Odorant binding proteins (OBPs) play an essential role for insect chemosensation in insect peripheral nervous systems of antennae. Each antennal sensilla contains more than one OBP at high concentrations but the interactions and cooperation between co-localized OBPs are rarely reported. In present study, we cloned, expressed and purified eight OBPs of the green peach aphid Myzus persicae. The effects of knocking down the expression of these OBP genes by RNAi on the electrophysiological and behavioural responses of M. persicae to the aphid alarm pheromone, (E)-ß-farnesene (EßF) were investigated. The results showed that the aphids could still be repelled by EßF when the expression of each of three OBP genes was individually knocked down. However, the simultaneous knockdown of MperOBP3/7/9 expression significantly reduced the electrophysiological response and the repellent behaviours of M. persicae to EßF than the single OBP gene knockdown (P < 0.05). Rather than a normal saturation binding curve of individual OBP, the binding curve of MperOBP3/7/9 is bell-shaped with a higher affinity for the fluorescent probe N-phenyl-1-naphthylamine (1-NPN). The competitive binding assays confirmed that MperOBP3, MperOBP7, MperOBP9 and MperOBP3/7/9 mixture exhibited a stronger binding affinity for EßF, than for sex pheromones and plant volatiles with a dissociation constant of 2.5 µM, 1.1 µM, 3.9 µM and 1.0 µM, respectively. The competitive binding curve of MperOBP3/7/9 mixture to EßF is shallow without bottom plateau, suggesting a conformational change and a rapid dissociation after the displacement of all 1-NPN (in vivo after the saturation binding of all OBPs by EßF). The interaction between OBPs and formation of a heterogeneous unit may facilitate the delivery EßF to the OR at electrophysiological and behavioural levels during insect odorant signal transduction thus mediate M. persicae response to the alarm pheromone EßF.

Transcriptional identification of differentially expressed genes associated with division of labor in Apis cerana cerana.

Workers of Apis cerana cerana undergo an in-hive nursing to outdoor foraging transition, but the genes underlying this age-related transition remain largely unknown. Here, we sequenced the head transcriptomes of its 7-day-old normal nurses, 18- and 22-day-old normal foragers, 7-day-old precocious foragers and 22-day-old over-aged nurses to unravel the genes associated with this transition. Mapping of the sequence reads to Apis mellifera genome showed that the three types of foragers had a greater percentage of reads from annotated exons and intergenic regions, whereas the two types of nurses had a greater percentage of reads from introns. Pair- and group-wise comparisons of the five transcriptomes revealed 59 uniquely expressed genes (18 in nurses and 41 in foragers) and 14 nurse- and 15 forager-upregulated genes. The uniquely expressed genes are usually low-abundance long noncoding RNAs, transcription factors, transcription coactivators, RNA-binding proteins, kinases or phosphatases that are involved in signaling and/or regulation, whereas the nurse- or forager-upregulated genes are often high-abundance downstream genes that directly perform the tasks of nurses or foragers. Taken together, these results suggest that the nurse-forager transition is coordinated by a social signal-triggered epigenetic shift from introns to exons/intergenic regions and the resulting transcriptional shift between the nurse- and forager-associated genes.

Broadly Tuned Odorant Receptor AlinOR59 Involved in Chemoreception of Floral Scent in Adelphocoris lineolatus.

Plant volatiles such as floral scent compounds play a crucial role in mediating insect host locating, mate search, and oviposition sites selection. The alfalfa plant bug, Adelphocoris lineolatus (Goeze), is a seriously polyphagous herbivore of alfalfa and cotton that has an obvious preference for flowering host plants. In this study, we focused on the role of an odorant receptor AlinOR59 in the perception of plant volatiles in A. lineolatus. In situ hybridization showed that AlinOR59 was coexpressed with the coreceptor AlinORco in the ORNs cell located in the long curved sensilla trichodea on antennae of both genders. The Xenopus oocytes expression coupled with two-electrode voltage clamp recordings demonstrated that AlinOR59 responded to 15 plant volatiles. In electroantennogram assays, all of the above 15 compounds could excite electrophysiological responses in the antennae of adult bugs. Furthermore, an important floral scent compound, methyl salicylate, was utilized to evaluate the behavioral responses of A. lineolatus. It was found that adult bugs of both genders were significantly attracted to methyl salicylate. Taken together, our findings suggest that AlinOR59 plays a crucial role in the perception of floral scents in A. lineolatus and could be used as a potential target to design novel olfactory regulators for the management of bugs.

Bioactivities of synthetic salicylate-substituted carboxyl (E)-ß-Farnesene derivatives as ecofriendly agrochemicals and their binding mechanism with potential targets in aphid olfactory system.

BACKGROUND: The aphid alarm pheromone, (E)-ß-farnesene (EßF), is a natural product secreted from the aphid cornicle as a signal to warn companions of danger. Odorant binding proteins (OBPs) are the vital targets in insect signal transduction pathways. To improve bioactivity of EßF as more economic and stable aphid control agents, EßF derivatives containing an active substructure, salicylic acid moiety, were designed, synthesized, and evaluated for their bioactivities in a structure-function study under laboratory conditions. RESULTS: EßF derivatives, (E)-3,7-dimethylocta-2,6-dien-1-yl-2-hydroxy-3-methylbenzoate and (E)-3,7-dimethylocta-2,6-dien-1-yl-2-hydroxy-3-methoxybenzoate showed outstanding aphid-repellent activity at a dose of 5 µg against Acyrthosiphon pisum (repellency proportions of 67.3% and 71.2%, respectively) and Myzus persicae (repellency proportions of 80.0% and 74.4%, respectively) in laboratory. EßF and most of its derivatives bound strongly to ApisOBP9 with a higher affinity than those of the reported potential targets AphisOBP3 and ApisOBP7. The binding affinities to these three ApisOBPs were generally consistent with the in vivo aphid-repellent activity. A molecular docking study suggested that the hydrophobic effect was crucial for the interactions between the derivatives and the OBPs. CONCLUSION: New EßF derivatives containing salicylic acid moiety and their repellent activity, binding mechanism with three potential OBPs are presented. A new OBP, ApisOBP9, was characterized as a potential EßF and EßF derivatives binding protein for the first time. The hydrophobic nature of these analogues is responsible for their activity. Two analogues 3b and 3e with outstanding aphid-repellent activity could be new leads for aphid control agents.

Seroprevalence of Bartonella henselae and Identification of Risk Factors in China.

Serum samples were tested for Bartonella henselae IgG antibodies using indirect immunofluorescence assays. We then analyzed associated risk factors. Serum samples were considered positive when reactive at a dilution of more than 1:320. Differences between groups and risk factors associated with Bartonella exposure were statistically analyzed using Chi-square tests and the generalized linear model. 122 of 1,260 samples (9.68%) were positive for B. henselae infection. The infection rate ranged from 0% to 30.43% and differed significantly among age groups ( P < 0.01); infection rate in the 50-59 years group was significantly higher than that in other age groups. The seroprevalence of Bartonella varied significantly among sites within the four provinces, and the infection rate of field workers was significantly higher than that of urban workers.

[Expression and characterizations of a novel full-length gene PKIbeta related to human glioma].

OBJECTIVE: To obtain differentially expressed genes related to human glioma using cDNA microarray and to characterization of one novel full-length gene. METHOD: Four samples of human glioma samples, 1 fetal brain tissue sample, and 2 normal brain tissue samples were used to extract the total RNA, and the mRNA was used to make probes. After hybridization and washing procedure, the products of hybridization were scanned using computer system. One gene, named 436F11 clone, was subsequently analyzed by Northern blotting, bioinformatics, and protein expression. RESULT: Fifteen differentially expressed new genes related to human glioma were obtained through four times of hybridization and scanning. Northern blotting confirmed that over-expression of 436F11 gene in the normal human brain tissue and low-expression in the human glioma tissues. The analysis of BLASTn and BLASTx showed that the clone of 436F11 was a novel full-length gene coding 78 amino acids of protein with a theoretical relative molecular weight of 8648 and an isoelectric point of 4.69 and that it was 60% identical to mouse PKIbeta amino acid, so it was called human PKIbeta gene. After it was transfected into Escherichia coli, higher-expressed protein of PKIbeta was obtained which yielded a major clear band on an SDS-PAGE gel after purification. The products obtained from amino acid sequencing and molecular weight detection were exactly the same as the products performed by bioinformatic analysis. CONCLUSION: cDNA microarray technology can be successfully applied to identify differentially expressed genes. PKIbeta may be a novel full-length gene related to human glioma and may provide a new way to gene therapy of glioma.

Expressional and functional comparisons of two general odorant binding proteins in Agrotis ipsilon.

Insect general odorant binding proteins (GOBPs) have been long thought to bind and transport host plant volatiles to the olfactory receptors on the dendrite membrane of the olfactory neurons. Recent studies indicate that they can also bind female sex pheromones. In present study, two GOBP genes, AipsGOBP1 and AipsGOBP2 were cloned from the adult antennae of Agrotis ipsilon. Tissue expression profiles indicated that both of them are antennae-specific and more abundant in the female antennae than in the male antennae. Temporal expression profiles showed that both AipsGOBP1 and AipsGOBP2 began to express in antennae 3 days prior to adult emergence from pupae, and reached their highest expression level 3 and 4 days after adult emergence, respectively. Mating increased their expression in the female antennae but reduced their expression in the male antennae. In situ hybridization and immunolocalization demonstrated that both AipsGOBP1 and AipsGOBP2 are expressed and co-localized in sensilla basiconica and sensilla trichodea of both sexes. AipsGOBP2 exhibited a high binding affinity in vitro with the two major sex pheromone components Z7-12:Ac and Z9-14:Ac and the four plant volatiles cis-3-hexen-1-ol, oleic acid, dibutyl phthalate and ß-caryophyllene with Ki values less than 5 µM. AipsGOBP1, on the other hand, showed medium binding affinities with the five A. ipsilon sex pheromones and six plant volatiles. AipsGOBP2 also showed a broader ligand-binding spectrum and a greater ligand-binding affinity than AipsGOBP1 with the tested aldehyde and alcohol sex pheromones of Lepidoptera species. Taken together, our results indicate that AipsGOBP2 may play greater roles than AipsGOBP1 does in binding sex pheromones and host plant volatiles.

A simpler and more cost-effective peptide biosynthetic method using the truncated GST as carrier for epitope mapping.

There is a need to develop better methods for epitope mapping and/or identification of antibody-recognizing motifs. Here, we describe improved biosynthetic peptide (BSP) method using a newly developed plasmid pXXGST-3 as vector, which has a viral E7 gene in the cloning sites of pXXGST-1. It is crucial to employ pXXGST-3 instead of pXXGST-1, since it makes use of the BSP method simpler and easier to perform, and more cost-effective for epitope mapping. These merits are embodied in two aspects: i) convenient recovery of double enzyme-digested product due to the existence of 315 bp inserted between BamH I and Sal I sites, and thus greatly reducing the production of self-ligation clones, and ii) no longer requiring control protein when screening recombinant (r-) clones expressing 8/18mer peptides by running polyacrylamide gel electrophoresis. The protocol involves the following core steps: (i) design of plus and minus strands of DNA fragments encoding overlapping 8/18mer peptides; (ii) chemical synthesis of the designed DNA fragments; (iii) development of r-clones using pXXGST-3 vector expressing each 8/18mer peptide fused with truncated GST188 protein; (iv) screening r-clones by running the cell pellets from each induced clone on SDS-PAGE gel followed by sequencing of inserted DNA fragments for each verified r-clone; and (v) Western blotting with either monoclonal antibodies or polyclonal antibodies. This improved GST188-BSP method provides a powerful alternative tool for epitope mapping.

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.

Odorant-binding and chemosensory proteins identified in the antennal transcriptome of Adelphocoris suturalis Jakovlev.

Adelphocoris suturalis Jakovlev (Hemiptera: Miridae) is an insect pest that causes severe agricultural damage to cotton and many other important crops. In insects, olfaction is very important throughout their lifetime. There are two groups of small soluble proteins, named odorant binding proteins (OBPs) and chemosensory proteins (CSPs), which are suggested to participate in the initial biochemical recognition steps of insect olfactory signal transduction. In this study, a total of 16 OBPs (12 classical OBPs and 4 plus-C OBPs) and 8 CSPs, were identified in the antennal transcriptome of A. suturalis. The sex- and tissue-specific profiles of these binding protein genes showed that 13 of the 16 OBP transcripts were highly expressed in the antennae of both sexes, and 4 OBPs (AsutOBP1, 4, 5 and 9) were expressed higher in the male antennae compared to the female antennae. Three CSPs (AsutCSP1, 4 and 5) were expressed specifically in the antennae of both sexes, and AsutCSP1 was expressed higher in the male antennae than in the female antennae. Our findings identify several novel OBP and CSP genes for further investigation of the olfactory system of A. suturalis at the molecular level.