'Candidatus Phytoplasma ziziphi' Changes the Metabolite Composition of Jujube Tree Leaves and Affects the Feeding Behavior of Its Insect Vector Hishimonus hamatus Kuoh.

Hishimonus hamatus Kuoh is a leafhopper species native to China that feeds on Chinese jujube leaves. This leafhopper species has been verified to transmit jujube witches' broom (JWB) disease, caused by phytoplasma, a fatal plant pathogen, which belongs to the phytoplasma subgroup 16SrV-B. The transmission of JWB phytoplasma largely relies on the feeding behavior of piercing-sucking leafhoppers. However, the specific mechanisms behind how and why the infection of JWB influences the feeding behavior of these leafhoppers are not fully understood. To address this, a study was conducted to compare the feeding patterns of H. hamatus when feeding JWB-infested jujube leaves to healthy leaves using the electrical penetration graph (EPG) technique. Then, a widely targeted metabolome analysis was performed to identify differences in the metabolite composition of JWB-infected jujube leaves and that of healthy jujube leaves. The results of EPG analyses revealed that when feeding on JWB-infected jujube leaves, H. hamatus exhibited an increased frequency of phloem ingestion and spent longer in the phloem feeding phase compared to when feeding on healthy leaves. In addition, the results of metabolomic analyses showed that JWB-infected leaves accumulated higher levels of small-molecular carbohydrates, free amino acids, and free fatty acids, as well as lower levels of lignans, coumarins and triterpenoids compared to healthy leaves. The above results indicated that the H. hamatus preferentially fed on the phloem of infected leaves, which seems to be linked to the transmission of the JWB phytoplasma. The results of metabolomic analyses partially imply that the chemical compounds might play a role in making the infected leaves more attractive to H. hamatus for feeding.

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

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

Different Binding Affinities of Three General Odorant-Binding Proteins in Grapholita funebrana (Treitscheke) (Lepidoptera: Tortricidae) to Sex Pheromones, Host Plant Volatiles, and Insecticides.

Insect general odorant-binding proteins (GOBPs) play irreplaceable roles in filtering, binding, and transporting host odorants to olfactory receptors. Grapholita funebrana (Treitscheke) (Lepidoptera: Tortricidae), an economically important pest of fruit crops, uses fruit volatiles as cues to locate host plants. However, the functions of GOBPs in G. funebrana are still unknown. Three GOBP genes, namely, GfunGOBP1, GfunGOBP2, and GfunGOBP3, were cloned, and their expression profiles in different tissues were detected by the method of real-time quantitative PCR (RT-qPCR). The binding properties of recombinant GfunGOBPs (rGfunGOBPs) to various ligands were investigated via fluorescence binding assays. The three GfunGOBPs were mainly expressed in the antennae of both male and female moths. All these three rGfunGOBPs could bind to sex pheromones, while having varying affinities toward these pheromones. The three rGfunGOBPs also displayed a wide range of ligand-binding spectrums with tested host odorants. The rGfunGOBP1, rGfunGOBP2, and rGfunGOBP3 bound to 34, 33, and 30 out of the 41 tested odorants, respectively. Three rGfunGOBPs had overlapping binding activities to ß-myrcene, (-)-α-phellandrene, and ethyl isovalerate with the Ki less than 3.0 µM. The rGfunGOBP1 and rGfunGOBP3 could selectively bind to several insecticides, whereas rGfunGOBP2 could not. Three rGfunGOBPs had the dual functions of selectively binding to sex pheromones and host odorants. Moreover, the rGfunGOBP1 and rGfunGOBP3 can also serve as 'signal proteins' and bind to different insecticides. This study contributed to elucidating the potential molecular mechanism of the olfaction for G. funebrana, and thereby promotes the development of effective botanical attractants or pheromone synergists to control G. funebrana.

Functional analysis of the odorant receptor coreceptor in odor detection in Grapholita molesta (lepidoptera: Tortricidae).

The olfactory system must detect and discriminate various semiochemicals in the environment. In response to such diversity, insects have evolved a family of odorant-gated ion channels composed of a common receptor (coreceptor, Orco) and a ligand-binding tuning odorant receptor (OR) that confers odour specificity. This study aims to examine the expression pattern of Orco gene of Grapholita molesta (GmolOrco) and to elucidate the role of GmolOrco in detecting G. molesta sex pheromone and green leaf volatiles by using gene silencing via RNA interference (RNAi) coupled antennal electrophysiological (EAG). Multiple sequence alignment showed that GmolOrco shared high sequence similarities with the Orco ortholog of lepidopterans. The results of real-time quantitative PCR detection demonstrated that GmolOrco was predominantly expressed in adult antennae and had the highest expression quantity in adult period among the different developmental stages. Compared with the noninjected controls, GmolOrco expression in GmolOrcodouble-stranded RNA (dsRNA)-injected males was reduced to 39.92% and that in females was reduced to 40.43%. EAG assays showed that the responses of GmolOrco-dsRNA injected males to sex pheromones (Z)-8-dodecenyl acetate (Z8-12:OAc) and (Z)-8-dodecenyl alcohol (Z8-12:OH) were significantly reduced, and the GmolOrco-dsRNA-injected female to green leaf volatile (Z)-3-hexenyl acetate also significantly declined. We inferred that Orco-mediated olfaction was different in male and female G. molesta adults and was mainly involved in recognizing the sex pheromones released by female moths.

Functional Analysis of the Chemosensory Protein GmolCSP8 From the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae).

Chemosensory proteins (CSPs) belong to a family of small water-soluble proteins that can selectively bind and transport odorant molecules for olfactory communication in insects. To date, their definite physiological functions in olfaction remain controversial when compared with odorant binding proteins (OBPs). To investigate the functions of CSPs in the oriental fruit moth Grapholita molesta, we determined the tissue expression patterns and binding properties of the CSP, GmolCSP8. The key binding sites of GmolCSP8 with a representative ligand were evaluated using molecular flexible docking, site-directed mutagenesis and ligand-binding experiments. Multiple sequence alignment and phylogenetic analysis showed that GmolCSP8 possesses a typical conserved four cysteines motif and shares high sequence identity with some CSP members of other Lepidopteran insects. GmolCSP8 was predominantly expressed in the wings and antennae of both male and female adults and may be involve in contact chemoreception. Recombinant GmolCSP8 (rGmolCSP8) exhibited specific-binding affinities to small aliphatic alcohols (C4-12) and had the strongest binding affinity to 1-hexanol. The three-dimensional structure of GmolCSP8 was constructed using the structure of sgCSP4 as a template. Site-directed mutagenesis and ligand-binding experiments confirmed that Thr27 is the key binding site in GmolCSP8 for 1-hexanol binding, because this residue can form hydrogen bond with the oxygen atom of the hydroxyl group in 1-hexanol, and Leu30 may play an important role in binding to 1-hexanol. We found that pH significantly affected the binding affinities of rGmolCSP8 to ligand, revealing that ligand-binding and -release by this protein is related to a pH-dependent conformational transition. Based on these results, we infer that GmolCSP8 may participate in the recognition and transportation of 1-hexanol and other small aliphatic alcohols.

Degradation of sex pheromone and plant volatile components by an antennal glutathione S-transferase in the oriental fruit moth,Grapholita molesta Busck (Lepidoptera: Tortricidae).

Insect antennae have a primary function of perceiving and discerning odorant molecules including sex pheromones and host plant volatiles. The assumption that genes highly expressed in the antennae may have an olfactory-related role associated with signal transduction. Here, one delta subfamily glutathione S-transferase (GST) gene (GmolGSTD1) was obtained from an antennal transcriptome of Grapholita molesta. Quantitative real-time polymerase chain reaction results revealed that GmolGSTD1 was mainly expressed in antennae and the expression levels were significantly higher in female antennae than in male antennae. The recombinant GmolGSTD1 (rGmolGSTD1) showed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The pH range for optimal rGmolGSTD1 enzyme activity was 6.0-6.5, and rGmolGSTD1 enzyme activity had maximal peaks at 35-40°C. Spectrophotometric analysis indicated that insecticides had weak inhibitory effects on the activity of rGmolGSTD1 with the inhibitory rates of 28.82% for chlorpyrifos, 22.27% for lambda-cyhalothrin, 18.07% for bifenthrin, 20.42% for acetamiprid, 17.57% for thiamethoxam, 25.67% for metaflumizone, 27.43% for abamectin, and 7.24% for chlorbenzuron. rGmolGSTD1 exhibited high degradation activity to the sex pheromone component (Z)-8-dodecenyl alcohol and the host plant volatile butyl hexanoate with the degradation efficiency of 75.01% and 48.54%, respectively. We speculate that GmolGSTD1 works in inactivating odorant molecules and maintaining sensitivity to olfactory communication of G. molesta.

Molecular and functional characterization of three odorant binding proteins from the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricide).

Odorant binding proteins (OBPs) act in recognizing odor molecules and their most well-studied functions are transporting odors across the sensillum lymph to olfactory receptor neurons within the insect antennal sensillum. The adults of Grapholita molesta highly depend on olfactory cues in locating host plants and selecting oviposition sites, in which OBPs play an important role in perceiving and recognizing host plant volatiles. Exploring the physiological function of OBPs could facilitate our understanding of their importance in insects' chemical communication. In this study, three OBP genes were cloned and named GmolOBP4, GmolOBP5, and GmolOBP10. Quantitative real-time PCR results indicated that GmolOBP4 and GmolOBP10 were predominantly expressed in adult antennae and GmolOBP5 was expressed in multiple tissues, including head, legs, and wings in addition to antennae. The binding affinities of the three recombinant GmolOBPs (rGmolOBPs) with four sex pheromone components and twenty-nine host plant volatiles were measured using 1-N-Phenyl-naphthylamine as a fluorescence probe. The three rGmolOBPs exhibited specific binding properties to potential ligands, GmolOBP4 and GmolOBP10 bound to minor sex pheromone components, such as (Z)-8-dodecenyl alcohol and dodecanol, respectively. rGmolOBP4 showed intermediate binding ability with hexanal, benzyl alcohol, and pear ester, rGmolOBP5 had a weak affinity for benzaldehyde, pear ester and, methyl jasmonate, and rGmolOBP10 showed strong binding capacity toward hexanol, decanol, and α-ocimene. We speculate that the GmolOBP4 and GmolOBP10 have dual functions in perception and recognition of host plant volatiles and sex pheromone components, while GmolOBP5 may serve other function(s).

Molecular and Functional Characterization of Odorant Binding Protein 7 From the Oriental Fruit Moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae).

Odorant-binding proteins (OBPs) are widely and abundantly distributed in the insect sensillar lymph and are essential for insect olfactory processes. The OBPs can capture and transfer odor molecules across the sensillum lymph to odorant receptors and trigger the signal transduction pathway. In this study, a putative OBP gene, GmolOBP7, was cloned using specific-primers, based on the annotated unigene which forms the antennal transcriptome of Grapholita molesta. Real-time PCR (qRT-PCR) analysis revealed that GmolOBP7 was highly expressed in the wings of males and the antennae of both male and female adult moths, while low levels were expressed in other tissues. The recombinant GmolOBP7 (rGmolOBP7) was successfully expressed and purified via Ni-ion affinity chromatography. The results of binding assays revealed that rGmolOBP7 exhibited a high binding affinity to the minor sex pheromone 1-dodecanol containing K i of 7.48 µM and had high binding capacities to the host-plant volatiles, such as pear ester, lauraldehyde and α-ocimene. RNA-interference experiments were performed to further assess the function of GmolOBP7. qRT-PCR showed that the levels of mRNA transcripts significantly declined in 1 and 2 day old male and female moths, treated with GmolOBP7 dsRNA, compared with non-injection controls. The EAG responses of dsRNA-injected males and females to pear ester, as well as the EAG responses of dsRNA-injected males to 1-dodecanol, were significantly reduced compared to the GFP-dsRNA-injected and non-injected controls. We therefore infer that GmolOBP7 has a dual function in the perception and recognition of the host-plant volatiles and sex pheromones.