Identification and functional analysis of odorant-binding proteins provide new control strategies for Apolygus lucorum.

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.

The microRNAs in the antennae of Apolygus lucorum (Hemiptera: Miridae): Expression properties and targets prediction.

MicroRNAs (miRNAs) regulate gene expression and contribute to numerous physiological processes. However, little is known about the functions of miRNAs in insect chemosensation. In this study, nine small RNA libraries were constructed and sequenced from the antennae of nymphs, adult males, and adult females of Apolygus lucorum. In total, 399 (275 known and 124 novel) miRNAs were identified. miR-7-5p_1 was the most abundant miRNA. Altogether, 69,708 target genes related to biogenesis, membrane, and binding activities were predicted. In particular, 15 miRNAs targeted 16 olfactory genes. Comparing the antennae of nymphs and adult males and females, 94 miRNAs were differentially expressed. Alternatively, a subset of differentially expressed miRNAs was verified by qPCR, supporting the reliability of the sequencing results. This study provides a global miRNA transcriptome for the antennae of A. lucorum and valuable information for further investigations of the functions of miRNAs in the regulation of chemosensation.

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.

Host plants transfer induced regulation of the chemosensory genes repertoire in the alfalfa plant bug Adelphocoris lineolatus (Goeze).

The alfalfa plant bug Adelphocoris lineolatus, an economically important pest, has representative behavioral characteristics with host plants transfer. Olfactory system is essential for insects to perceive ever-changing chemical signals in the external environment, and chemosensory genes play crucial roles in signals reception and transduction. In this work, we compared the differences in chemosensory genes expression before and after host plants transfer by constructing 12 antennal transcriptomes of male and female bugs, respectively. The results showed that the expression levels of most chemosensory genes in A. lineolatus changed to adapt to the transformation of the hosts plant. More remarkable, female bugs had more up-regulated chemosensory genes than males. Differentially expressed genes (DEGs) analysis revealed three odorant binding proteins (OBPs), three chemosensory proteins (CSPs), eight odorant receptors (ORs) and one ionotropic receptor (IR) showed significant differences when the host plant transferred. There were complex characteristics of up- and down- regulated genes in male and female adults, among which OBP19 showed higher expression in females exposing to the new host plant alfalfa, suggesting this OBP may be associated with the localization of the oviposition site. The OR54 and OR82 were up-regulated in both genders, indicating their possible roles in recognizing some alfalfa-specific volatiles. These findings will provide valuable insights in biological functions of chemosensory genes in A. lineolatus and facilitate the development of new targets for novel strategies to control the alfalfa plant bug and other herbivores.

Engineering Nicotiana tabacum for the de novo biosynthesis of DMNT to regulate orientation behavior of the parasitoid wasps Microplitis mediator.

BACKGROUND: (E)-4,8-dimethylnona-1,3,7-triene (DMNT), one of the homoterpenes, is thought to contribute to plant indirect defense against insect herbivores. DMNT-enriched plants have great application potential to regulate insect behavior in the 'push & pull' strategy of pest management. However, de novo biosynthesis of DMNT in plants without a homoterpene metabolic pathway in their wild type is still not achieved, and the role of DMNT played in these plants and their interacted insects remains unclear. RESULTS: Cytochrome P450s and terpene synthases involved in homoterpenes biosynthesis in cotton plants were employed to generate DMNT-releasing tobacco plants. Single GhTPS14 transgenic Nicotiana tabacum only emitted (E)-nerolidol, the precursor of DMNT. Transgenic tobaccos expressing single GhCYP82Ls were unable to produce DMNT or TMTT, while DMNT was detected when exogenous (E)-nerolidol was added. Compared to wild-type plants, only co-expression of GhCYP82Ls and GhTPS14 in transgenic tobaccos triggered the constitutive release of single-component DMNT. Furthermore, DMNT-emitting transgenic tobacco plants, whether infested with Helicoverpa armigera larvae or not, significantly incited orientation behavior of parasitoid wasps Microplitis mediator. CONCLUSION: Wild type N. tabacum plants have no DMNT metabolic pathway. DMNT could be de novo biosynthesized via co-expression of GhCYP82Ls and GhTPS14. What is more, the parasitoid wasp M. mediator could be recruited by DMNT-releasing transgenic tobaccos, especially by H. armigera-infested transgenic tobaccos, suggesting the potential roles of engineered N. tabacum in regulating the behavioral preference of M. mediator.

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.

Full-length transcriptome analysis of Phytolacca americana and its congener P. icosandra and gene expression normalization in three Phytolaccaceae species.

BACKGROUND: Phytolaccaceae species in China are not only ornamental plants but also perennial herbs that are closely related to human health. However, both large-scale full-length cDNA sequencing and reference gene validation of Phytolaccaceae members are still lacking. Therefore, single-molecule real-time sequencing technology was employed to generate full-length transcriptome in invasive Phytolacca americana and non-invasive exotic P. icosandra. Based on the transcriptome data, RT-qPCR was employed to evaluate the gene expression stability in the two plant species and another indigenous congener P. acinosa. RESULTS: Total of 19.96 Gb and 19.75 Gb clean reads of P. americana and P. icosandra were generated, including 200,857 and 208,865 full length non-chimeric (FLNC) reads, respectively. Transcript clustering analysis of FLNC reads identified 89,082 and 98,448 consensus isoforms, including 86,989 and 96,764 high-quality ones. After removing redundant reads, 46,369 and 50,220 transcripts were obtained. Based on structure analysis, total 1675 and 1908 alternative splicing variants, 25,641 and 31,800 simple sequence repeats (SSR) as well as 34,971 and 36,841 complete coding sequences were detected separately. Furthermore, 3574 and 3833 lncRNA were predicted and 41,676 and 45,050 transcripts were annotated respectively. Subsequently, seven reference genes in the two plant species and a native species P. acinosa were selected and evaluated by RT-qPCR for gene expression analysis. When tested in different tissues (leaves, stems, roots and flowers), 18S rRNA showed the highest stability in P. americana, whether infested by Spodoptera litura or not. EF2 had the most stable expression in P. icosandra, while EF1-α was the most appropriate one when attacked by S. litura. EF1-α showed the highest stability in P.acinosa, whereas GAPDH was recommended when infested by S. litura. Moreover, EF1-α was the most stable one among the three plant species whenever germinating seeds or flowers only were considered. CONCLUSION: Full-length transcriptome of P. americana and P. icosandra were produced individually. Based on the transcriptome data, the expression stability of seven candidate reference genes under different experimental conditions was evaluated. These results would facilitate further exploration of functional and comparative genomic studies in Phytolaccaceae and provide insights into invasion success of P. americana.

Identification of Leg Chemosensory Genes and Sensilla in the Apolygus lucorum.

Apolygus lucorum (Hemiptera: Miridae), one of the main insect pests, causes severe damage in cotton and many other economic crops. As is well-known, legs play important roles in the chemoreception of insects. In this study, the putative chemosensory proteins in legs of A. lucorum involved in close or contact chemical communication of adult bugs were investigated using RNA transcriptome sequencing and qPCR methods. Transcriptome data of forelegs, middle legs and hind legs of adult bugs demonstrated that 20 odorant binding protein (OBP) genes, eight chemosensory protein (CSP) genes, one odorant receptor (OR) gene, one ionotropic receptor (IR) gene and one sensory neuron membrane protein (SNMP) gene were identified in legs of A. lucorum. Compared to the previous antennae transcriptome data, five CSPs, IR21a and SNMP2a were newly identified in legs. Results of qPCR analysis indicated that all these putative chemosensory genes were ubiquitously expressed in forelegs, middle legs and hind legs of bugs. Furthermore, four types of sensilla on legs of A. lucorum including sensilla trichodea (subtypes: long straight sensilla trichodea, Str1; long curved sensilla trichodea, Str2), sensilla chaetica (subtypes: sensilla chaetica 1, Sch1; sensilla chaetica 2, Sch2; and sensilla chaetica 3, Sch3), sensilla basiconca (subtypes: medium-long sensilla basiconca, Sba1; short sensilla basiconca, Sba2) and Böhm bristles (BB) were found using scanning electron microscopy. Additionally, the largest number of sensilla was observed on hind legs, while the forelegs had the smallest number of sensilla. Our data provide valuable insights into understanding the chemoreception of legs in A. lucorum.

Functional characterization of one sex pheromone receptor (AlucOR4) in Apolygus lucorum (Meyer-Dür).

Traps baited with female-produced sex pheromones have been very effective in the monitoring and management of mirid bugs in numerous field trials. However, none of the target odorant receptors for sex pheromone components in Apolygus lucorum have been identified. Here, we identified one candidate sex pheromone receptor, AlucOR4, from A. lucorum. Quantitative real-time PCR (qPCR) analysis revealed that AlucOR4 was antennae-enriched and male-biased in adult A. lucorum. Xenopus oocyte expression system assays demonstrated that AlucOR4/AlucOrco was sensitive to two major sex pheromone constituents and exhibited high sensitivity to (E)-2-hexenyl butyrate (E2HB) and lower sensitivity to hexyl butyrate (HB). The expression level of target mRNA was significantly reduced (>80%) in dsAlucOR4-injected bugs after five days. The electroantennogram (EAG) responses of male antennae to E2HB and HB were also reduced significantly (~40%). Our findings suggest that AlucOR4 is essential to sex pheromone perception in A. lucorum.

Deorphanization of an odorant receptor revealed new bioactive components for green mirid bug Apolygus lucorum (Hemiptera: Miridae).

BACKGROUND: The Apolygus lucorum is one of the most destructive insect pests in China with a wide range of host plants. Interaction of A. lucorum with surrounding environment heavily relies on chemical communication. Deorphanization of receptors involved in odors detection elevates our understanding of the olfactory system of this pest and may help to develop a chemical ecology-based control strategy. RESULTS: AlucOR80, an odorant receptor (OR) in A. lucorum was newly cloned. Gene expression analysis showed that this receptor was mainly expressed in the antennae and head of both sexes but with a male bias. The Xenopus oocytes heterologous expression system coupled with the two-electrode voltage-clamp (TEVC) recording revealed that AlucOR80 was tuned to 21 selected compounds. Furthermore, electroantennogram (EAG) tests confirmed that all 21 ligands of AlucOR80 were electrophysiologically active in antennae of both sexes. Behavioral trials in a three-cage olfactometer indicated that 16 compounds were behaviorally active, amongst which, 12 components were attractants and four components were repellents for adults of both sexes. Butyl butyrate and Dimethyl disulfide (DMDS) were the strongest attractive and repellant compounds, respectively. Importantly, we found the repellency of 1, 8-Cineole, S-(-)-cis-Verbenol and (1S)-(1)-beta-Pinene against adults of A. lucorum. CONCLUSION: Although AlucOR80 is a general OR, may play important role in the olfactory perception of A. lucorum. Screening of AlucOR80 ligands by behavioral assay provided valuable insights by which olfactory-based management approaches could be developed by utilizing the behaviorally active components as attractants or repellents. © 2019 Society of Chemical Industry.

Mouthparts enriched odorant binding protein AfasOBP11 plays a role in the gustatory perception of Adelphocoris fasciaticollis.

Insect odorant binding proteins (OBPs), one of the most important groups of odor carriers, are believed to play essential roles in chemoreception. In the present study, we focused on AfasOBP11 in Adelphocoris fasciaticollis. Expression profiles showed that AfasOBP11 was mainly expressed in the mouthparts of A. fasciaticollis. Additionally, two types of sensilla, sensilla trichodeum and sensilla basiconicum, were found on the mouthparts of bugs. Moreover, anti-AfasOBP11 antiserum strongly labeled the sensilla basiconica. In fluorescence binding assays, recombinant AfasOBP11 displayed much stronger binding abilities to non-volatile secondary metabolite compounds than to volatile odors, suggesting a role of AfasOBP11 in taste sensing. To further investigate the biological functions of AfasOBP11, the feeding behavior of wild-type, dsGFP-injected and dsAfasOBP11-injected bugs was evaluated by performing electrical penetration graph (EPG) tests. After RNA interference of target AfasOBP11, A. fasciaticollis bugs spent a longer time and pierced more frequently on the artificial diet containing 2.0% gossypol, indicating that RNAi treated bugs reduced sensitivity to gossypol. Our findings suggest that AfasOBP11 may play a vital role in chemoreception of A. fasciaticollis, especially in gustatory perception.

Identification and functional analysis of two P450 enzymes of Gossypium hirsutum involved in DMNT and TMTT biosynthesis.

The homoterpenes (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) are major herbivore-induced plant volatiles that can attract predatory or parasitic arthropods to protect injured plants from herbivore attack. In this study, DMNT and TMTT were confirmed to be emitted from cotton (Gossypium hirsutum) plants infested with chewing caterpillars or sucking bugs. Two CYP genes (GhCYP82L1 and GhCYP82L2) involved in homoterpene biosynthesis in G. hirsutum were newly identified and characterized. Yeast recombinant expression and enzyme assays indicated that the two GhCYP82Ls are both responsible for the conversion of (E)-nerolidol to DMNT and (E,E)-geranyllinalool to TMTT. The two heterologously expressed proteins without cytochrome P450 reductase fail to convert the substrates to homoterpenes. Quantitative real-time PCR (qPCR) analysis suggested that the two GhCYP82L genes were significantly up-regulated in leaves and stems of G. hirsutum after herbivore attack. Subsequently, electroantennogram recordings showed that electroantennal responses of Microplitis mediator and Peristenus spretus to DMNT and TMTT were both dose dependent. Laboratory behavioural bioassays showed that females of both wasp species responded positively to DMNT and males and females of M. mediator could be attracted by TMTT. The results provide a better understanding of homoterpene biosynthesis in G. hirsutum and of the potential influence of homoterpenes on the behaviour of natural enemies, which lay a foundation to study genetically modified homoterpene biosynthesis and its possible application in agricultural pest control.

Identification and expression analysis of an olfactory receptor gene family in green plant bug Apolygus lucorum (Meyer-Dür).

Olfactory receptors are believed to play a central role in insects host-seeking, mating, and ovipositing. On the basis of male and female antennal transcriptome of adult Apolygus lucorum, a total of 110 candidate A. lucorum odorant receptors (AlucOR) were identified in this study including five previously annotated AlucORs. All the sequences were validated by cloning and sequencing. Tissue expression profiles analysis by RT-PCR indicated most AlucORs were antennal highly expressed genes. The qPCR measurements further revealed 40 AlucORs were significantly higher in the antennae. One AlucOR was primarily expressed in the female antennae, while nine AlucORs exhibited male-biased expression patterns. Additionally, both the RPKM value and RT-qPCR analysis showed AlucOR83 and AlucOR21 were much higher abundant in male antennae than in female antennae, suggesting their different roles in chemoreception of gender. Phylogenetic analysis of ORs from several Hemipteran species demonstrated that most AlucORs had orthologous genes, and five AlucOR-specific clades were defined. In addition, a sub-clade of potential male-based sex pheromone receptors were also identified in the phylogenetic tree of AlucORs. Our results will facilitate the functional studies of AlucORs, and thereby provide a foundation for novel pest management approaches based on these genes.

Transcriptomic and Expression Analysis of the Salivary Glands in White-Backed Planthoppers, Sogatella furcifera.

The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is one of the serious rice pests because of its destructive feeding. The salivary glands of the WBPH play an important role in the feeding behaviour. Currently, however, very little is known about the salivary glands at the molecular level. We sequenced the salivary gland transcriptome (sialotranscripome) of adult WBPHs using the Illumina sequencing. A total of 65,595 transcripts and 51,842 unigenes were obtained from salivary glands. According to annotations against the Nr database, many of the unigenes identified were associated with the most studied enzymes in hemipteran saliva. In the present study, we identified 32 salivary protein genes from the WBPH sialotranscripome, which were categorized as those involved in sugar metabolism, detoxification, suppression of plant defense responses, immunity-related responses, general digestion, and other phytophagy processes. Tissue expression profiles analysis revealed that four of 32 salivary protein genes (multicopper oxidase 4, multicopper oxidase 6, carboxylesterase and uridine phosphorylase 1 isform X2) were primarily expressed in the salivary gland, suggesting that they played putative role in insect-rice interactions. 13 of 32 salivary protein genes were primarily expressed in gut, which might play putative role in digestive and detoxify mechanism. Development expression profiles analysis revealed that the expression level of 26 of 32 salivary protein genes had no significant difference, suggesting that they may play roles in every developmental stages of salivary gland of WBPH. The other six genes have a high expression level in the salivary gland of adult. 31 of 32 genes (except putative acetylcholinesterase 1) have no significant difference in male and female adult, suggesting that their expression level have no difference between sexes. This report analysis of the sialotranscripome for the WBPH, and the transcriptome provides a foundational list of the genes involved in feeding. Our data will be useful to investigate the mechanisms of interaction between the WBPH and the host plant.

Reference Gene Selection and Evaluation for Gene Expression Studies Using qRT-PCR in the White-Backed Planthopper, Sogatella furcifera (Hemiptera: Delphacidae).

The white-backed planthopper, Sogatella furcifera (Hemiptera, Delphacidae), is one of the most devastating rice pests. For a better control strategy, various genetic studies have been conducted using reverse-transcription quantitative real-time polymerase chain reaction (qRT-PCR). The appropriate application of qRT-PCR requires reliable endogenous controls; however, studies on this aspect of the white-backed planthopper are lacking. In the present study, nine commonly used reference genes, elongation factor 1-α (EF1-α), polyubiquitin (UB), ribosomal protein S18 (RPS18), actin 1 (ACT), α-1 tubulin (TUB), glyceraldehyde-3-phosphate (GAPDH), ribosomal protein L9 (RPL9), ribosomal protein L10 (RPL10), and 18S ribosomal RNA (18S), were evaluated by qRT-PCR for their expression stability under four different experimental conditions (different developmental stages, acquisition of Southern rice black-streaked dwarf virus (SRBSDV), different tissues, and different temperature stress). These results were analyzed using four software programs (geNorm, NormFinder, BestKeeper, and the delta Ct method) and a Web-based comprehensive tool RefFinder to compare and rank candidate reference genes. According to the results of RefFinder analysis, which integrates the abovementioned four software programs, TUB was ranked as the most suitable reference gene at different developmental stages and under different temperature stress, and GAPDH and RPL9 showed the highest stability for acquisition of SRBSDV and different tissues, respectively. These results will provide a solid foundation for future gene expression study on the white-backed planthopper, and also will give aids in establishing a standardized qRT-PCR procedure for other related insects.

Relation Between the Viral Load Accumulation of Southern Rice Black-Streaked Dwarf Virus and the Different Developmental Stages of Sogatella furcifera (Hemiptera: Delphacidae).

The white-backed planthopper, Sogatella furcifera (Horvath), is currently the only confirmed vector of Southern rice black-streaked dwarf virus (SRBSDV), which causes severe rice production losses in China. In this study, an absolute quantification qPCR method was used to detect viral gene mRNA expression levels at different developmental stages of white-backed planthoppers fed SRBSDV-infected rice plants. A comparison of viral copy numbers of the SRBSDV S10 gene at the same developmental stage indicated that the white-backed planthopper had higher viral copy numbers when the virus was acquired at the earlier developmental stages. The adult-stage white-backed planthoppers that had acquired the virus at the first-second nymphal stage displayed significantly higher viral titers than white-backed planthoppers that acquired the virus at the third-fourth nymphal stage, at the fifth nymphal stage, and at the adult stage. The fifth nymphal stage white-backed planthoppers that acquired the virus at the first-second nymphal stage displayed higher viral copy numbers than fifth nymphal stage white-backed planthoppers that acquired the virus at the third-fourth nymphal stage and at the fifth nymphal stage. The highest viral load value appeared in the middle adult stage. The annual immigration characteristics of white-backed planthoppers would be beneficial for the dispersal of SRBSDV because this virus could be transmitted far away following the migration of vigorous planthoppers. Therefore, investigating the change in the viral load at different life stages of SRBSDV-positive individuals is required to develop more effective control of the spread of SRBSDV in the field.