Antennal Transcriptome Analysis of Olfactory Genes and Characterization of Odorant Binding Proteins in Odontothrips loti (Thysanoptera: Thripidae).

To identify odors in complex environments accurately, insects have evolved multiple olfactory proteins. In our study, various olfactory proteins of Odontothrips loti Haliday, an oligophagous pest that primarily affects Medicago sativa (alfalfa), were explored. Specifically, 47 putative olfactory candidate genes were identified in the antennae transcriptome of O. loti, including seven odorant-binding proteins (OBPs), nine chemosensory proteins (CSPs), seven sensory neuron membrane proteins (SNMPs), eight odorant receptors (ORs), and sixteen ionotropic receptors (IRs). PCR analysis further confirmed that 43 out of 47 genes existed in O. loti adults, and O.lotOBP1, O.lotOBP4, and O.lotOBP6 were specifically expressed in the antennae with a male-biased expression pattern. In addition, both the fluorescence competitive binding assay and molecular docking showed that p-Menth-8-en-2-one, a component of the volatiles of the host, had strong binding ability to the O.lotOBP6 protein. Behavioral experiments showed that this component has a significant attraction to both female and male adults, indicating that O.lotOBP6 plays a role in host location. Furthermore, molecular docking reveals potential active sites in O.lotOBP6 that interact with most of the tested volatiles. Our results provide insights into the mechanism of O. loti odor-evoked behavior and the development of a highly specific and sustainable approach for thrip management.

Stemborer-induced rice plant volatiles boost direct and indirect resistance in neighboring plants.

Herbivore-induced plant volatiles (HIPVs) are known to be perceived by neighboring plants, resulting in induction or priming of chemical defenses. There is little information on the defense responses that are triggered by these plant-plant interactions, and the phenomenon has rarely been studied in rice. Using chemical and molecular analyses in combination with insect behavioral and performance experiments, we studied how volatiles emitted by rice plants infested by the striped stemborer (SSB) Chilo suppressalis affect defenses against this pest in conspecific plants. Compared with rice plants exposed to the volatiles from uninfested plants, plants exposed to SSB-induced volatiles showed enhanced direct and indirect resistance to SSB. When subjected to caterpillar damage, the HIPV-exposed plants showed increased expression of jasmonic acid (JA) signaling genes, resulting in JA accumulation and higher levels of defensive proteinase inhibitors. Moreover, plants exposed to SSB-induced volatiles emitted larger amounts of inducible volatiles and were more attractive to the parasitoid Cotesia chilonis. By unraveling the factors involved in HIPV-mediated defense priming in rice, we reveal a key defensive role for proteinase inhibitors. These findings pave the way for novel rice management strategies to enhance the plant's resistance to one of its most devastating pests.

Identification of Odorant-Binding and Chemosensory Protein Genes in Mythimna separata Adult Brains Using Transcriptome Analyses.

Large numbers of chemosensory genes have been identified in the peripheral sensory organs of the pest Mythimna separata (Walker) to increase our understanding of chemoreception-related molecular mechanisms and to identify molecular targets for pest control. Chemosensory-related genes are expressed in various tissues, including non-sensory organs, and they play diverse roles. To better understand the functions of chemosensory-related genes in non-sensory organs, transcriptomic analyses of M. separata brains were performed. In total, 29 odorant-binding proteins (OBPs) and 16 chemosensory proteins (CSPs) putative genes were identified in the transcriptomic data set. The further examination of sex- and tissue-specific expression using RT-PCR suggested that eight OBPs (OBP5, -7, -11, -13, -16, -18, -21, and -24) and eight CSPs (CSP2-4, -8, CSP10-12, and -15) genes were expressed in the brain. Furthermore, bands representing most OBPs and CSPs could be detected in antennae, except for a few that underwent sex-biased expression in abdomens, legs, or wings. An RT-qPCR analysis of the expression profiles of six OBPs (OBP3-5, -9, -10, and -16) and two CSPs (CSP3 and CSP4) in different tissues and sexes indicated that OBP16 was highly expressed in male brain, and CSP3 and CSP4 were female-biased and highly expressed in brain. The expression levels of OBP5 and OBP10 in brain were not significantly different between the sexes. The findings expand our current understanding of the expression patterns of OBPs and CSPs in M. separata sensory and non-sensory tissues. These results provide valuable reference data for exploring novel functions of OBPs and CSPs in M. separata and may help in developing effective biological control strategies for managing this pest by exploring novel molecular targets.

Consumption of miRNA-Mediated Insect-Resistant Transgenic Rice Pollen Does Not Harm Apis mellifera Adults.

RNAi-based insect-resistant genetically engineered (IRGE) crops represent a promising approach for pest management by suppressing gene expressions or translation. A developed microRNA-mediated IRGE rice line expressing endogenous Chilo suppressalis Csu-novel-260 shows significant resistance to target pests. The nontarget insect Apis mellifera is an important pollinator used as a surrogate species for the ecological risk assessment of IRGE plants. To simulate a worst-case scenario, the full-length C. suppressalis and A. mellifera disembodied (dib) cDNAs were cloned. The dib 3'-untranslated regions shared 58.06% nucleotide sequence similarity between C. suppressalis and A. mellifera. No potential Csu-novel-260 binding site in Amdib was detected through the bioinformatics analysis. A dietary RNAi toxicity assay of the impacts of ingested Csu-novel-260 on A. mellifera adults showed that the survival rates of RNAi-treated A. mellifera did not significantly differ from those in the blank control (CK) and negative control (NC) treatments. The Csu-novel-260 uptake by A. mellifera peaked at 8 days postfeeding and then gradually decreased. The Amdib expression was not affected by the RNAi assay days or treatments. These results suggest that A. mellifera adults are not susceptible to high doses of Csu-novel-260 in the dietary RNAi assay and that the impact of miRNA-mediated IRGE plants on A. mellifera is negligible.

The overexpression of insect endogenous microRNA in transgenic rice inhibits the pupation of Chilo suppressalis and Cnaphalocrocis medinalis.

BACKGROUND: Chilo suppressalis and Cnaphalocrocis medinalis are destructive rice pests co-occurring in major rice-growing areas in China. RNA interference (RNAi)-based insect-resistant genetically engineered (IRGE) crops provide a promising approach for pest management by suppressing gene expression or translation. A microRNA (miRNA)-mediated IRGE rice line expressing endogenous Chilo suppressalis miRNA Csu-novel-260, showing significant resistance against Chilo suppressalis, provides an attractive control strategy for Chilo suppressalis by suppressing the expression of the disembodied (dib) gene expression. However, whether this transgenic line also shows the resistance against Cnaphalocrocis medinalis remains unknown. RESULTS: A spatiotemporal expression analysis of Csu-novel-260 in the transgenic rice line was performed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) to determine the paddy field pest exposure dose. In diet feeding assays, a chemically synthesized Csu-novel-260 agomir at 200 fmol g-1 significantly inhibited Chilo suppressalis pupation. However, larval development, survival and pupal weight were not significantly affected. Additionally, the transgenic line significantly affected Cnaphalocrocis medinalis pupation but not larval survival. The qRT-PCR showed that Csdib and Cmdib expression levels were significantly suppressed when the two pests fed on the transgenic line. Additionally, the transgenic line significantly decreased Cry1C-resistant and Cry1C-susceptible Chilo suppressalis larval survival in detached rice tissue feeding assays, indicating that Cry1C-resistant Chilo suppressalis was not cross-resistant to Csu-novel-260 expressed in miRNA-mediated IRGE rice. CONCLUSION: Our study demonstrated that miRNA-mediated IRGE rice significantly inhibited Chilo suppressalis and Cnaphalocrocis medinalis pupation. The results provide a new viewpoint for the application of RNAi-based plants and the inspiration for environmental risk assessment.

Reduced Mythimna separata infestation on Bt corn could benefit aphids.

Use of genetically engineered plants that express insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) have been proven efficacious for managing lepidopteran pests. However, in some cases herbivores that are not targeted by the Bt trait have increased in importance. It has been suggested that reduced caterpillar damage to Bt crops could lead to decreased levels of induced plant defensive compounds which might benefit other non-target herbivores. Here we investigated the potential effect of reduced damage by larvae of Mythimna separata on aphid populations in Bt corn. We compared the performance of Rhopalosiphum maidis feeding on non-Bt corn plants that had been infested by M. separata larvae or were uninfested. The results showed that caterpillar-infested corn plants significantly reduced the fitness of R. maidis leading to a prolonged nymphal development time, reduced adult longevity and fecundity compared to uninfested plants. Consequently, the population growth rate of corn aphids feeding on caterpillar-infested corn plants was significantly lower than on uninfested plants. As expected, the aphids performed significantly better on Lepidoptera-resistant Bt corn than on non-Bt corn when plants were infested with M. separata, since the caterpillars caused very little damage to the Bt plants. The current findings indicate that reduced M. separata infestation could benefit aphid development in Bt corn. Bt corn has the potential to be commercialized in China in the near future and aphids and other non-target pests should be monitored in the farming fields.

Odorant Receptors for Detecting Flowering Plant Cues Are Functionally Conserved across Moths and Butterflies.

Odorant receptors (ORs) are essential for plant-insect interactions. However, despite the global impacts of Lepidoptera (moths and butterflies) as major herbivores and pollinators, little functional data are available about Lepidoptera ORs involved in plant-volatile detection. Here, we initially characterized the plant-volatile-sensing function(s) of 44 ORs from the cotton bollworm Helicoverpa armigera, and subsequently conducted a large-scale comparative analysis that establishes how most orthologous ORs have functionally diverged among closely related species whereas some rare ORs are functionally conserved. Specifically, our systematic analysis of H. armigera ORs cataloged the wide functional scope of the H. armigera OR repertoire, and also showed that HarmOR42 and its Spodoptera littoralis ortholog are functionally conserved. Pursuing this, we characterized the HarmOR42-orthologous ORs from 11 species across the Glossata suborder and confirmed the HarmOR42 orthologs form a unique OR lineage that has undergone strong purifying selection in Glossata species and whose members are tuned with strong specificity to phenylacetaldehyde, a floral scent component common to most angiosperms. In vivo studies via HarmOR42 knockout support that HarmOR42-related ORs are essential for host-detection by sensing phenylacetaldehyde. Our work also supports that these ORs coevolved with the tube-like proboscis, and has maintained functional stability throughout the long-term coexistence of Lepidoptera with angiosperms. Thus, beyond providing a rich empirical resource for delineating the precise functions of H. armigera ORs, our results enable a comparative analysis of insect ORs that have apparently facilitated and currently sustain the intimate adaptations and ecological interactions among nectar feeding insects and flowering plants.

Transcriptomic and Metabolomic Responses of Rice Plants to Cnaphalocrocis medinalis Caterpillar Infestation.

Interactions between plants and insect herbivores are important determinants of plant productivity in cultivated and natural agricultural fields. The rice leaf folder (Cnaphalocrocis medinalis) causes tremendous damage to rice production in Asian countries. However, little information is available about how rice plants defend themselves against this destructive pest at molecular and biochemical levels. Here, we observed the transcriptomic and metabolomic differences in rice leaves after 0, 1, 6, 12, and 24 h of being fed by C. medinalis using RNA sequencing and metabolome profiling. Transcriptional analyses showed that gene expression responds rapidly to leaf folder infestation, with the most significant transcriptional changes occurring within 6 h after the initiation of feeding. Metabolite abundance changed more slowly than gene expression. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the rice transcriptional response to infestation involved genes encoding protein kinases, transcription factors, biosynthesis of secondary metabolites, photosynthesis, and phytohormone signaling. Moreover, the jasmonic acid-dependent signaling pathway triggered by leaf folder herbivory played a vital role in rice defense against this pest. Taken together, our results provide comprehensive insights into the defense system of rice to this species and may inform the development of insect-resistant rice varieties.

Identification and sex-biased profiles of candidate olfactory genes in the antennal transcriptome of the parasitoid wasp Cotesia vestalis.

Cotesia vestalis (Hymenoptera: Braconidae) is an endoparasitoid wasp that attacks larvae of the diamondback moth, Plutella xylostella (Linnaeus), one of the most important pests of cruciferous plants in the world. This natural enemy has been used to control populations of P. xylostella. To more fully understand the molecular mechanisms of olfactory reception in C. vestalis, the transcriptomes of male antennae, female antennae, and mixed female and male legs were systematically analyzed, and the chemosensory genes were identified. A total of 253 olfactory genes, including candidates of 158 odorant receptors (ORs), 12 ionotropic receptors (IRs), 48 gustatory receptors (GRs), 22 odorant binding proteins (OBPs), 11 chemosensory proteins (CSPs), and two sensory neuron membrane proteins (SNMPs), were systematically identified and subjected to sequence and phylogenetic analysis. Then, the differentially expressed genes (DEGs) within female and male antennae were analyzed to obtain expression information on the candidate olfactory genes at the transcriptional level. The expression levels of 25 ORs, nine OBPs, eight GRs, and three IRs were significantly different between male and female antennae. Our results provide valuable information for further studies on the chemoreception mechanisms in C. vestalis, to ultimately improve the pest control measures by using natural enemies.

Cadherin CsCad plays differential functional roles in Cry1Ab and Cry1C intoxication in Chilo suppressalis.

Transgenic rice lines expressing Bacillus thuringiensis (Bt) toxins have been successfully developed for the control of Chilo suppressalis. However, the evolution of insect resistance is a major threat to Bt rice durability. Bt toxins function by binding specific receptors in the midgut of target insects; specifically, cadherin proteins have been identified as Cry toxin receptors in diverse lepidopteran species. Here, we report the functional roles of cadherin CsCad in the midgut of C. suppressalis in Cry1Ab and Cry1C toxicity. We expressed a recombinant truncated CsCad peptide (CsCad-CR11-MPED) in Escherichia coli that included the eleventh cadherin repeat and MPED region. Based on ligand blotting and ELISA binding assays, the CsCad-CR11-MPED peptide specifically bound Cry1Ab with high affinity but weakly bound Cry1C. The CsCad-CR11-MPED peptide significantly enhanced the susceptibility of C. suppressalis larvae to Cry1Ab but not Cry1C. Furthermore, the knockdown of endogenous CsCad with Stealth siRNA reduced C. suppressalis larval susceptibility to Cry1Ab but not Cry1C, suggesting that CsCad plays differential functional roles in Cry1Ab and Cry1C intoxication in C. suppressalis. This information directly enhances our understanding of the potential resistance mechanisms of C. suppressalis against Bt toxins and may assist in the development of effective strategies for delaying insect resistance.

Functional Analysis of MsepOR13 in the Oriental Armyworm Mythimna separata (Walker).

Olfaction in insects has a critical role in recognizing the host, finding food, and choosing mating partners, as well as avoiding predators. Odorant receptors (ORs), which are housed in the dendritic membrane of sensory neurons and extended into the lymph of sensilla on insect antennae, are participating in the detection of volatile compounds in insects. In the present study, we identified an OR gene, named MsepOR13, in the oriental armyworm Mythimna separata (Walker). Quantitative real-time polymerase chain reaction revealed that MsepOR13 was expressed mainly in the antennae of male and female moths. In in vitro heterologous expression experiments, MsepOR13 was widely tuned to 32 of the 67 different compounds tested. Furthermore, MsepOR13 responded to eugenol at a low concentration of 10-9 M, with an EC50 value of 3.91 × 10-6 M. The high sensitivity suggests an important role for the OR13 gene in the moth olfactory system.

Functional characterization of four sex pheromone receptors in the newly discovered maize pest Athetis lepigone.

Chemoreception systems play a crucial role in regulating key behavioral activities of insects, such as mating, oviposition, and foraging. Odorant receptors (ORs) trigger the transduction of chemical signals into electric signals, and are involved in the corresponding responses associated with odorant guidance behaviors. Pheromone receptors (PRs) of male adult insects are generally thought to function in the recognition of female sex pheromones, and are also important molecular targets for the development of behavioral inhibitors and insecticides. In this study, we successfully expressed and functionally analyzed four AlepPRs of Athetis lepigone in Xenopus oocytes using the two-electrode voltage-clamp method. The results demonstrated that AlepOR3 responded exclusively to the sex pheromone compound of A. lepigone, (Z)-7-dodecenyl acetate (Z7-12:Ac) (EC50 = 8.830 × 10-6 M), while AlepOR4 responded to all five compounds [(Z7-12:Ac, (Z)-8-dodecenyl acetate (Z8-12:Ac), (Z)-9-tetradecenyl acetate (Z9-14:Ac), (Z,E)-9,11-tetradecadienyl acetate (Z9,E11-14:Ac), and (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:Ac)] and had a higher response to Z9-14:Ac (EC50 = 2.243 × 10-5 M) than to Z7-12:Ac. However, AlepOR6 displayed a significantly higher response to a non-pheromone of A. lepigone, Z9,E12-14:Ac (EC50 = 7.145 × 10-6 M), than to the other four compounds. AlepOR5 displayed no responses to any of the pheromone compounds of A. lepigone, but responded exclusively to (Z)-11-hexadecenyl acetate (Z11-16:Ac) (EC50 = 7.870 × 10-6 M), a sex pheromone compound of other Noctuidae species. These findings can help explore the molecular mechanisms of sex pheromone recognition in A. lepigone and other moths, and develop broad-spectrum behavioral inhibitors and insecticides against different maize moths in future.

Identification of candidate chemosensory genes in Mythimna separata by transcriptomic analysis.

BACKGROUND: The oriental armyworm, Mythimna separata, is an economically important and common Lepidopteran pest of cereal crops. Chemoreception plays a key role in insect life, such as foraging, oviposition site selection, and mating partners. To better understand the chemosensory mechanisms in M. separata, transcriptomic analysis of antennae, labial palps, and proboscises were conducted using next-generation sequencing technology to identify members of the major chemosensory related genes. RESULTS: In this study, 62 putative odorant receptors (OR), 20 ionotropic receptors (IR), 16 gustatory receptors (GR), 38 odorant binding proteins (OBP), 26 chemosensory proteins (CSP), and 2 sensory neuron membrane proteins (SNMP) were identified in M. separata by bioinformatics analysis. Phylogenetic analysis of these candidate proteins was performed. Differentially expressed genes (DEGs) analysis was used to determine the expressions of all candidate chemosensory genes and then the expression profiles of the three families of receptor genes were confirmed by real-time quantitative RT-PCR (qPCR). CONCLUSIONS: The important genes for chemoreception have now been identified in M. separata. This study will provide valuable information for further functional studies of chemoreception mechanisms in this important agricultural pest.

Identification and characterization of chemosensory genes in the antennal transcriptome of Spodoptera exigua.

Chemical senses are crucial for insect behaviors such as host preference, mate choice and oviposition site selection. Various protein families are involved in these processes, including odorant receptors (ORs), ionotropic receptors (IRs), gustatory receptors (GRs), chemosensory proteins (CSPs), odorant binding proteins (OBPs) and sensory neuron membrane proteins (SNMPs). To better understand the olfactory mechanism in Spodoptera exigua, we conducted transcriptome analysis of adult antennae and identified a total of 157 candidate chemosensory genes encoding 51 ORs, 20 IRs, 7 GRs, 32 CSPs, 45 OBPs and 2 SNMPs. Quantitative real time PCR (qPCR) analysis of the tissue- and sex-specific expression profiles of ORs, GRs and IRs revealed that these genes could be detected in at least one tissue tested. SexiOR6, 11, 13 and 16, which were predicted to be pheromone receptors based on phylogenetic analysis, exhibited male-specific antennae expression. SexiOR18, 26, 28, 30, 34, 39, and 40 exhibited female-biased expression. SexiGR1, SexiGR2 and SexiGR3, are predicted carbon dioxide receptors, and the former was expressed specifically in antennae, and the latter two were expressed both in antennae and labial palps. SexiIRs had diverse expression profiles. SexiIR8a and SexiIR25a were quite conserved and expressed at high levels. This work will greatly facilitate the understanding of olfactory system in S. exigua and provides valuable information for further functional studies of the chemoreception mechanism in Lepidopteran moths.