Overexpression of Two Odorant Binding Proteins Confers Chlorpyrifos Resistance in the Green Peach Aphid Myzus persicae.

The green peach aphid, Myzus persicae, is a worldwide agricultural pest. Chlorpyrifos has been widely used to control M. persicae for decades, thus leading to a high resistance to chlorpyrifos. Recent studies have found that insect odorant binding proteins (OBPs) play essential roles in insecticide resistance. However, the potential resistance mechanism underlying the cross-link between aphid OBPs and chlorpyrifos remains unclear. In this study, two OBPs (MperOBP3 and MperOBP7) were found overexpressed in M. persicae chlorpyrifos-resistant strains (CRR) compared to chlorpyrifos-sensitive strains (CSS); furthermore, chlorpyrifos can significantly induce the expression of both OBPs. An in vitro binding assay indicated that both OBPs strongly bind with chlorpyrifos; an in vivo RNAi and toxicity bioassay confirmed silencing either of the two OBPs can increase the susceptibility of aphids to chlorpyrifos, suggesting that overexpression of MperOBP3 and MperOBP7 contributes to the development of resistance of M. persicae to chlorpyrifos. Our findings provide novel insights into insect OBPs-mediated resistance mechanisms.

The role of OR5, which is highly expressed in the winged grain aphid Sitobion miscanthi, in specific recognition of EBF.

Winged parthenogenetic aphids are mainly responsible for migration and dispersal. Aphid alarm pheromone (E)-ß-Farnesene (EBF) has dual effects on repelling and stimulating wing differentiation in aphids. Previous studies have shown that the odorant coreceptor SmisOrco is involved in the perception of EBF by S. miscanthi; however, its EBF-specific odorant receptor (OR) and the difference between winged and wingless aphids remain unclear. In this study, the Xenopus oocyte expression system and RNAi technology were used to detect the transmission of EBF signals, and it was found that the olfactory receptor SmisOR5 is an EBF-specific OR in S. miscanthi and is specifically highly expressed in the antennae of winged aphids. Furthermore, when OR5 was silenced with dsRNA, the repellent effect of EBF was weakened, and aphids showed more active aimless movements. Therefore, as a specific OR for EBF, the high expression level of SmisOR5 in winged aphids suggests a molecular basis for its high sensitivity to EBF. This study advances our understanding of the molecular mechanisms of aphid EBF perception and provides novel ideas for effective management and prevention of the migration of winged aphids.

The ATP-binding cassette transporter subfamily G member 4 mediates cuticular hydrocarbon transport to regulate drought tolerance in Acyrthosiphon pisum.

ABC transporters are a highly conserved membrane protein class that promote the transport of substances across membranes. Under drought conditions, insects primarily regulate the content of cuticular hydrocarbon (CHC) to retain water and prevent evaporative loss. Involvement of ABC transporter protein G (ABCG) subfamily genes in insect CHC transport has been relatively understudied. In this study, we demonstrated that ABCG4 gene in Acyrthosiphon pisum (ApABCG4) is involved in CHC transport and affects drought tolerance by regulating CHC accumulation. ApABCG4 is strongly expressed in the abdominal cuticle and embryonic stages of A. pisum. Effective silencing of ApABCG4 was achieved using RNAi, and the silencing duration was analyzed. ApABCG4 silencing resulted in a significant decrease in the total and component contents of the CHC and cuticular waxy coatings of A. pisum. Nevertheless, the internal hydrocarbon content remained unchanged. The lack of cuticular hydrocarbons significantly reduced the drought tolerance of A. pisum, shortening its survival time under drought stress. Drought stress caused significant upregulation of ApABCG4. Molecular docking showed that ApABCG4 has a high binding affinity for nine n-alkanes of CHC through electrostatic interactions. These results indicate that ApABCG4 is a novel RNAi target with key applications in aphid biological control.

microRNA maintains nutrient homeostasis in the symbiont-host interaction.

Endosymbionts provide essential nutrients for hosts, promoting growth, development, and reproduction. However, the molecular regulation of nutrient transport from endosymbiont to host is not well understood. Here, we used bioinformatic analysis, RNA-Sequencing, luciferase assays, RNA immunoprecipitation, and in situ hybridization to show that a bacteriocyte-distributed MRP4 gene (multidrug resistance-associated protein 4) is negatively regulated by a host (aphid)-specific microRNA (miR-3024). Targeted metabolomics, microbiome analysis, vitamin B6 (VB6) supplements, 3D modeling/molecular docking, in vitro binding assays (voltage clamp recording and microscale thermophoresis), and functional complementation of Escherichia coli were jointly used to show that the miR-3024/MRP4 axis controls endosymbiont (Serratia)-produced VB6 transport to the host. The supplementation of miR-3024 increased the mortality of aphids, but partial rescue was achieved by providing an external source of VB6. The use of miR-3024 as part of a sustainable aphid pest-control strategy was evaluated by safety assessments in nontarget organisms (pollinators, predators, and entomopathogenic fungi) using virus-induced gene silencing assays and the expression of miR-3024 in transgenic tobacco. The supplementation of miR-3024 suppresses MRP4 expression, restricting the number of membrane channels, inhibiting VB6 transport, and ultimately killing the host. Under aphids facing stress conditions, the endosymbiont titer is decreased, and the VB6 production is also down-regulated, while the aphid's autonomous inhibition of miR-3024 enhances the expression of MRP4 and then increases the VB6 transport which finally ensures the VB6 homeostasis. The results confirm that miR-3024 regulates nutrient transport in the endosymbiont-host system and is a suitable target for sustainable pest control.

Composition and abundance of midgut plasma membrane proteins in two major hemipteran vectors of plant viruses, Bemisia tabaci and Myzus persicae.

Multiple species within the order Hemiptera cause severe agricultural losses on a global scale. Aphids and whiteflies are of particular importance due to their role as vectors for hundreds of plant viruses, many of which enter the insect via the gut. To facilitate the identification of novel targets for disruption of plant virus transmission, we compared the relative abundance and composition of the gut plasma membrane proteomes of adult Bemisia tabaci (Hemiptera: Aleyrodidae) and Myzus persicae (Hemiptera: Aphididae), representing the first study comparing the gut plasma membrane proteomes of two different insect species. Brush border membrane vesicles were prepared from dissected guts, and proteins extracted, identified and quantified from triplicate samples via timsTOF mass spectrometry. A total of 1699 B. tabaci and 1175 M. persicae proteins were identified. Following bioinformatics analysis and manual curation, 151 B. tabaci and 115 M. persicae proteins were predicted to localize to the plasma membrane of the gut microvilli. These proteins were further categorized based on molecular function and biological process according to Gene Ontology terms. The most abundant gut plasma membrane proteins were identified. The ten plasma membrane proteins that differed in abundance between the two insect species were associated with the terms "protein binding" and "viral processes." In addition to providing insight into the gut physiology of hemipteran insects, these gut plasma membrane proteomes provide context for appropriate identification of plant virus receptors based on a combination of bioinformatic prediction and protein localization on the surface of the insect gut.

Dynamic response of essential amino acid biosynthesis in Buchnera aphidicola to supplement sub-optimal host nutrition.

The endosymbiotic bacterium Buchnera aphidicola allows its host Acyrthosiphon pisum to utilise a nutritionally limited phloem sap diet without significant mortality by providing essential amino acids (EAAs), which it biosynthesises de novo via complex pathways consisting of multiple enzymes. Previous studies have reported how non-essential amino acids (NEAAs) provided by the host are utilised by B. aphidicola, along with how genes within the biosynthetic pathways respond to amino acid deficiency. Although the effect on B. aphidicola gene expression upon the removal of a single EAA and multiple NEAAs from the A. pisum diet has been reported, little is known about the effects of the complete simultaneous removal of multiple EAAs, especially branched-chain amino acids (BCAAs). To investigate this, A. pisum was provided with amino acid deficient diets ilv- (lacking isoleucine, leucine, valine) or thra- (lacking threonine, methionine, lysine). Due to their involvement in the production of several amino acids, the expression of genes ilvC, ilvD (both involved in isoleucine, leucine and valine biosynthesis) and thrA (involved in threonine, methionine and lysine biosynthesis) was analysed and the expression of trpC (involved in tryptophan biosynthesis) was used as a control. Survival was reduced significantly when A. pisum was reared on ilv- or thra- (P < 0.001 and P = 0.000 respectively) compared to optimal artificial diet and was significantly lower on ilv- (P < 0.001) than thra-. This is likely attributed to the EAAs absent from ilv- being required at higher concentrations for aphid growth, than those EAAs absent from thra-. Expression of ilvC and ilvD were upregulated 2.49- and 2.08-fold (respectively) and thrA expression increased 2.35- and 2.12-fold when A. pisum was reared on ilv- and thra- (respectively). The surprisingly large upregulation of thrA when reared on ilv- is likely due to threonine being an intermediate in isoleucine biosynthesis. Expression of trpC was not affected by rearing on either of the two amino acid deficient diets. To our knowledge this study has shown, for the first time, how genes within the biosynthetic pathways of an endosymbiont respond to the simultaneous complete omission of multiple EAAs as well as all three BCAAs (leucine, isoleucine, valine), from the host diet.

Salivary gland transcriptomics of the cotton aphid Aphis gossypii and comparative analysis with other sap-sucking insects.

Aphids are sap-sucking insects responsible for crop losses and a severe threat to crop production. Proteins in the aphid saliva are integral in establishing an interaction between aphids and plants and are responsible for host plant adaptation. The cotton aphid, Aphis gossypii (Hemiptera: Aphididae) is a major pest of Gossypium hirsutum. Despite extensive studies of the salivary proteins of various aphid species, the components of A. gossypii salivary glands are unknown. In this study, we identified 123,008 transcripts from the salivary gland of A. gossypii. Among those, 2933 proteins have signal peptides with no transmembrane domain known to be secreted from the cell upon feeding. The transcriptome includes proteins with more comprehensive functions such as digestion, detoxification, regulating host defenses, regulation of salivary glands, and a large set of uncharacterized proteins. Comparative analysis of salivary proteins of different aphids and other insects with A. gossypii revealed that 183 and 88 orthologous clusters were common in the Aphididae and non-Aphididae groups, respectively. The structure prediction for highly expressed salivary proteins indicated that most possess an intrinsically disordered region. These results provide valuable reference data for exploring novel functions of salivary proteins in A. gossypii with their host interactions. The identified proteins may help develop a sustainable way to manage aphid pests.

Flavonols do not affect aphid load in green or senescing birch leaves but coincide with a decrease in Photosystem II functionality.

Instead of red anthocyanins, birches synthesise colourless (to human eye), UV-absorbing flavonols during autumn senescence. To test if flavonols protect against insects, and if leaves with high or low amounts of flavonols differ in their photosynthetic functions, aphid-free and aphid-infested green and senescing birch leaves were collected from outdoor-grown trees and analysed. Photosynthetic parameters were greatly affected by the leaf chlorophyll content (i.e. the phase of senescence). Photochemical quenching and the amount of functional Photosystem I decreased linearly with chlorophyll content, while FV/FM (Photosystem II functionality) decreased strongly only at the end of senescence. Non-photochemical quenching of excitation energy (NPQ) increased towards the end of senescence. However, no significant differences in the total flavonol amounts, nor in individual flavonol species, were found between aphid-free and aphid-infested leaves, suggesting that flavonols play no role in defence against aphid herbivory. Interestingly, both green and senescing leaves with a high flavonol content showed low FV/FM values. High flavonol content slowed down PSII photoinhibition and improved recovery, but only in green leaves. Previously, we proposed that anthocyanins provide an additional sink for photosynthates at the nitrogen resorption phase during autumn senescence, and the present data may suggest that flavonol synthesis plays a similar role.

Coupling clearing and hybridization chain reaction approaches to investigate gene expression in organs inside intact insect heads.

Detecting RNA molecules within their natural environment inside intact arthropods has long been challenging, particularly in small organisms covered by a tanned and pigmented cuticle. Here, we have developed a methodology that enables high-resolution analysis of the spatial distribution of transcripts of interest without having to dissect tiny organs or tissues, thereby preserving their integrity. We have combined an in situ amplification approach based on hybridization chain reaction, which enhances the signal-to-noise ratio, and a clearing approach that allows the visualization of inner organs beneath the cuticle. We have implemented this methodology for the first time in Hemiptera, mapping two salivary aphid (Acyrthosiphon pisum) transcripts, the effector c002 and the salivary sheath protein SHP. With a multiplex approach, we could simultaneously detect different mRNAs in mounted pea aphid head-thorax samples and show that they were distributed in distinct secretory cells of salivary glands. RESEARCH HIGHLIGHTS: Combining hybridisation chain reaction and clearing allows the detection of transcripts in intact aphids heads. The transcripts of the two salivary proteins c002 and SHP are compartmentalized in distinct secretory cells of the principal glands.

Molecular mechanisms for selective action of afidopyropen to Myzus persicae and Coccinella septempunctata.

BACKGROUND: Afidopyropen is a novel insecticide with high selectivity between sucking insects such as the peach aphids Myzus persicae and natural enemies like the seven-spotted lady beetle Coccinella septempunctata. However, the mechanisms of selective action for afidopyropen remain unknown. RESULTS: The LC50 values of afidopyropen to the 1st-4th instar larvae and adult C. septempunctata were 372- to more than 7267-fold higher than that to adult M. persicae. Though the activity of cytochrome P450s in M. persicae was 6.1- to 7.5-fold higher than that in C. septempunctata, the latter has much higher activities of carboxylesterase (CarEs) and glutathione S-transferases (GSTs), and the crude enzyme of C. septempunctata and M. persicae showed similar metabolism efficiency to afidopyropen. Molecular docking results demonstrated that afdopyropen showed higher binding affinity to the vanilloid-type transient receptor potential (TRPV) channel of M. persicae (-9.1 kcal/mol) than to that of C. septempunctata (-8.2 kcal/mol). And the EC50 value of afdopyropen to the TRPV channel of C. septempunctata (41 360 nM) was 19 885-fold higher than that in M. persicae (2.08 nM). CONCLUSIONS: Our results demonstrated that the significantly different sensitivity of M. persicae and C. septempunctata TRPV channel to afidopyropen play a key role in the high selectivity of afidopyropen. These findings provide new insights into the selective mechanisms of afidopyropen against insect pests and natural enemies as well as the theory support for coordinated application of chemical control and biological control. © 2024 Society of Chemical Industry.

Mesoporous silica nanospheres-mediated insecticide and antibiotics co-delivery system for synergizing insecticidal toxicity and reducing environmental risk of insecticide.

Mesoporous silica nanoparticles (MSNs) are efficient carriers of drugs, and are promising in developing novel pesticide formulations. The cotton aphids Aphis gossypii Glover is a world devastating insect pest. It has evolved high level resistance to various insecticides thus resulted in the application of higher doses of insecticides, which raised environmental risk. In this study, the MSNs based pesticide/antibiotic delivery system was constructed for co-delivery of ampicillin (Amp) and imidacloprid (IMI). The IMI@Amp@MSNs complexes have improved toxicity against cotton aphids, and reduced acute toxicity to zebrafish. From the 16S rDNA sequencing results, Amp@MSNs, prepared by loading ampicillin to the mesoporous of MSNs, greatly disturbed the gut community of cotton aphids. Then, the relative expression of at least 25 cytochrome P450 genes of A. gossypii was significantly suppressed, including CYP6CY19 and CYP6CY22, which were found to be associated with imidacloprid resistance by RNAi. The bioassay results indicated that the synergy ratio of ampicillin to imidacloprid was 1.6, while Amp@MSNs improved the toxicity of imidacloprid by 2.4-fold. In addition, IMI@Amp@MSNs significantly improved the penetration of imidacloprid, and contributed to the amount of imidacloprid delivered to A. gossypii increased 1.4-fold. Thus, through inhibiting the relative expression of cytochrome P450 genes and improving penetration of imidacloprid, the toxicity of IMI@Amp@MSNs was 6.0-fold higher than that of imidacloprid. The greenhouse experiments further demonstrated the enhanced insecticidal activity of IMI@Amp@MSNs to A. gossypii. Meanwhile, the LC50 of IMI@Amp@MSNs to zebrafish was 3.9-fold higher than that of IMI, and the EC50 for malformation was 2.8-fold higher than IMI, respectively, which indicated that the IMI@Amp@MSNs complexes significantly reduced the environmental risk of imidacloprid. These findings encouraged the development of pesticide/antibiotic co-delivery nanoparticles, which would benefit pesticide reduction and environmental safety.

Molecular and functional characterization of heat-shock protein 70 in Aphis gossypii under thermal and xenobiotic stresses.

Aphis gossypii, a globally distributed and economically significant pest of several crops, is known to infest a wide range of host plants. Heat shock proteins (Hsps), acting as molecular chaperones, are essential for the insect's environmental stress responses. The present study investigated the molecular characteristics and expression patterns of AgHsp70, a heat shock protein gene, in Aphis gossypii. Our phylogenetic analysis revealed that AgHsp70 shared high similarity with homologs from other insects, suggesting a conserved function across species. The developmental expression profiles of AgHsp70 in A. gossypii showed that the highest transcript levels were observed in the fourth instar nymphs, while the lowest levels were detected in the third instar nymphs. Heat stress and exposure to four different xenobiotics (2-tridecanone, tannic acid, gossypol, and flupyradifurone (4-[(2,2-difluoroethyl)amino]-2(5H)-furanone)) significantly up-regulated AgHsp70 expression. Knockdown of AgHsp70 using RNAi obviously increased the susceptibility of cotton aphids to 2-tridecanone, gossypol and flupyradifurone. Dual-luciferase reporter assays revealed that gossypol and flupyradifurone significantly enhanced the promoter activity of AgHsp70 at a concentration of 10 mg/L. Furthermore, we identified the transcription factor heat shock factor (HSF) as a regulator of AgHsp70, as silencing AgHSF reduced AgHsp70 expression. Our results shed light on the role of AgHsp70 in xenobiotic adaptation and thermo-tolerance.

CF2-II Alternative Splicing Isoform Regulates the Expression of Xenobiotic Tolerance-Related Cytochrome P450 CYP6CY22 in Aphis gossypii Glover.

The expression of P450 genes is regulated by trans-regulatory factors or cis-regulatory elements and influences how endogenous or xenobiotic substances are metabolized in an organism's tissues. In this study, we showed that overexpression of the cytochrome P450 gene, CYP6CY22, led to resistance to cyantraniliprole in Aphis gossypii. The expression of CYP6CY22 increased in the midgut and remaining carcass of the CyR strain, and after repressing the expression of CYP6CY22, the mortality of cotton aphids increased 2.08-fold after exposure to cyantraniliprole. Drosophila ectopically expressing CYP6CY22 exhibited tolerance to cyantraniliprole and cross-tolerance to xanthotoxin, quercetin, 2-tridecanone, tannic acid, and nicotine. Moreover, transcription factor CF2-II (XM_027994540.2) is transcribed only as the splicing variant isoform CF2-II-AS, which was found to be 504 nucleotides shorter than CF2-II in A. gossypii. RNAi and yeast one-hybrid (Y1H) results indicated that CF2-II-AS positively regulates CYP6CY22 and binds to cis-acting element p (-851/-842) of CYP6CY22 to regulate its overexpression. The above results indicated that CYP6CY22 was regulated by the splicing isoform CF2-II-AS, which will help us further understand the mechanism of transcriptional adaption of cross-tolerance between synthetic insecticides and plant secondary metabolites mediated by P450s.

Host-specific co-evolution likely driven by diet in Buchnera aphidicola.

BACKGROUND: Russian wheat aphid (Diuraphis noxia Kurd.) is a severe pest to wheat, and even though resistance varieties are available to curb this pest, they are becoming obsolete with the development of new virulent aphid populations. Unlike many other aphids, D noxia only harbours a single endosymbiont, Buchnera aphidicola. Considering the importance of Buchnera, this study aimed to elucidate commonalities and dissimilarities between various hosts, to better understand its distinctiveness within its symbiotic relationship with D. noxia. To do so, the genome of the D. noxia's Buchnera was assembled and compared to those of other aphid species that feed on diverse host species. RESULTS: The overall importance of several features such as gene length and percentage GC content was found to be critical for the maintenance of Buchnera genes when compared to their closest free-living relative, Escherichia coli. Buchnera protein coding genes were found to have percentage GC contents that tended towards a mean of ~ 26% which had strong correlation to their identity to their E. coli homologs. Several SNPs were identified between different aphid populations and multiple isolates of Buchnera were confirmed in single aphids. CONCLUSIONS: Establishing the strong correlation of percentage GC content of protein coding genes and gene identity will allow for identifying which genes will be lost in the continually shrinking Buchnera genome. This is also the first report of a parthenogenically reproducing aphid that hosts multiple Buchnera strains in a single aphid, raising questions regarding the benefits of maintaining multiple strains. We also found preliminary evidence for post-transcriptional regulation of Buchnera genes in the form of polyadenylation.

Discovery of Potential Neonicotinoid Insecticides by an Artificial Intelligence Generative Model and Structure-Based Virtual Screening.

The identification of neonicotinoid insecticides bearing novel scaffolds is of great importance for pesticide discovery. Here, artificial intelligence-based tools and virtual screening strategy were integrated to discover potential leads of neonicotinoid insecticides. A deep generative model was successfully constructed using a recurrent neural network combined with transfer learning. The model evaluation showed that the pretrained model could accurately grasp the SMILES grammar of drug-like molecules and generate potential neonicotinoid compounds after transfer learning. The generated molecules were evaluated by hierarchical virtual screening, hits were subjected to a similarity search, and the most similar structures were purchased for the bioassay. Compounds A2 and A5 displayed 52.5 and 50.3% mortality rates against Aphis craccivora at 100 mg/L, respectively. The docking study indicated that these two compounds have similar binding modes to neonicotinoids, which were verified by further molecular dynamics simulations.

Use of double-stranded RNA targeting ß2 divergent nicotinic acetylcholine receptor subunit to control pea aphid Acyrthosiphon pisum at larval and adult stages.

BACKGROUND: In recent years, the use of the RNA interference technology (RNAi) has emerged as one of the new strategies for species-specific control of insect pests. Its specificity depends on the distinctiveness of the target gene sequence for a given species. In this work, we assessed in the pea aphid Acyrthosiphon pisum (A. pisum) the use of a double-stranded RNA (dsRNA) that targets the ß2 divergent nicotinic acetylcholine receptor (nAChR) subunit (dsRNA-ß2), which shares low sequence identity with other subunits, to control populations of this pest at different developmental stages. Because nAChRs are targeted by neonicotinoid insecticides such as imidacloprid, we also assessed the effect of dsRNA-ß2 coupled to this insecticide on aphid survival. Finally, because the effect of a control agent on beneficial insect must be considered before any use of new pest management strategies, the acute toxicity of dsRNA-ß2 combined with imidacloprid was evaluated on honeybee Apis mellifera. RESULTS: In this work, we demonstrated that dsRNA-ß2 alone has an insecticidal effect on aphid larvae and adults. Moreover, dsRNA-ß2 and imidacloprid effects on aphid larvae and adults were additive, meaning that dsRNA-ß2 did not alter the efficacy of imidacloprid on these two developmental stages. Also, no obvious acute toxicity on Apis mellifera was reported. CONCLUSION: Using RNAi that targets ß2 divergent nAChR subunit is effective alone or combined with imidacloprid to control A. pisum at larval and adult stages. Because no obvious Apis mellifera mortality has been reported, this RNAi-based pest management strategy should be considered to control insect pest. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Identification of salivary proteins of the cowpea aphid Aphis craccivora by transcriptome and LC-MS/MS analyses.

Aphid salivary proteins mediate the interaction between aphids and their host plants. Moreover, these proteins facilitate digestion, detoxification of secondary metabolites, as well as activation and suppression of plant defenses. The cowpea aphid, Aphis craccivora, is an important sucking pest of leguminous crops worldwide. Although aphid saliva plays an important role in aphid plant interactions, knowledge of the cowpea aphid salivary proteins is limited. In this study, we performed transcriptomic and LC-MS/MS analyses to identify the proteins present in the salivary glands and saliva of A. craccivora. A total of 1,08,275 assembled transcripts were identified in the salivary glands of aphids. Of all these assembled transcripts, 53,714 (49.11%) and 53,577 (49.48%) transcripts showed high similarity to known proteins in the Nr and UniProt databases, respectively. A total of 2159 proteins were predicted as secretory proteins from the salivary gland transcriptome dataset, which contain digestive enzymes, detoxification enzymes, previously known effectors and elicitors, and potential proteins whose functions have yet to be determined. The proteomic analysis of aphid saliva resulted in the identification of 171 proteins. Tissue-specific expression of selected genes using RT-PCR showed that three genes were expressed only in the salivary glands. Overall, our results provide a comprehensive repertoire of cowpea aphid salivary proteins from the salivary gland and saliva, which will be a good resource for future effector functional studies and might also be useful for sustainable aphid management.

Fusion dsRNA in targeting salivary protein genes enhance the RNAi-based aphid control.

RNA interference (RNAi) is a promising tool for pest control and relies on sequence-specific gene silencing. Salivary proteins are cooperatively secreted into plants to guarantee the feeding of aphids; thus they have potential to develop as selective targets for RNAi-based pest control strategy. For this purpose, we firstly analyzed 18 salivary proteomes of various aphid species, and these salivary proteins can be mainly categorized into seven functional groups. Secondly, we created a work-flow for fusion dsRNA design that can target multiple genes but were selectively safe to beneficial insects. Based on this approach, seven fusion dsRNAs were designed to feed the green peach aphid, which induced a significant reduction in aphid fitness. Among them, ingestion of dsperoxidase induced the highest mortality in aphids, which was also significantly higher than that of traditional dsRNAs in targeting three peroxidases separately. In addition, dsperoxidase-fed green peach aphids triggered the highest H2O2 content of host plants as well as the attraction to natural enemies (ladybeetle and parasitic wasp) but repellent to other control aphids. Our results indicate that the fusion dsRNA design approach can improve aphid control capacity, and the fusion dsRNA targeting salivary protein-encoding genes can enhance the direct and indirect defenses of host plants, thus providing a new strategy for RNAi-based aphid control.

Development of Sustainable Insecticide Candidates for Protecting Pollinators: Insight into the Bioactivities, Selective Mechanism of Action and QSAR of Natural Coumarin Derivatives against Aphids.

Plants employ abundant toxic secondary metabolites to withstand insect attack, while pollinators can tolerate some natural defensive compounds. Coumarins, as promising green alternatives to chemical insecticides, possess wide application prospects in the crop protection field. Herein, the bioactivities of 30 natural coumarin derivatives against Aphis gossypii were assessed and revealed that 6-methylcoumarin exhibited potent aphicidal activity against aphids but displayed no toxicity to honeybees. Additionally, using biochemical, bioinformatic, and molecular assays, we confirmed that the action mode of 6-methylcoumarin against aphids was by inhibiting acetylcholinesterase (AChE). Meanwhile, functional assays revealed that the difference in action site, which located in Lys585 in aphid AChE (equivalent to Val548 in honeybee AChE), was the principal reason for 6-methylcoumarin being toxic to aphids but safe to pollinators. This action site was further validated by mutagenesis data, which uncovered how 6-methylcoumarin was unique selective to the aphid over honeybee or mammalian AChE. Furthermore, a 2D-QSAR model was established, revealing that the central structural feature was H3m, which offers guidance for the future design of more potent coumarin compounds. This work provides a sustainable strategy to take advantage of coumarin analogues for pest management while protecting nontarget pollinators.

Horizontally Transferred Salivary Protein Promotes Insect Feeding by Suppressing Ferredoxin-Mediated Plant Defenses.

Herbivorous insects such as whiteflies, planthoppers, and aphids secrete abundant orphan proteins to facilitate feeding. Yet, how these genes are recruited and evolve to mediate plant-insect interaction remains unknown. In this study, we report a horizontal gene transfer (HGT) event from fungi to an ancestor of Aleyrodidae insects approximately 42 to 190 million years ago. BtFTSP1 is a salivary protein that is secreted into host plants during Bemisia tabaci feeding. It targets a defensive ferredoxin 1 in Nicotiana tabacum (NtFD1) and disrupts the NtFD1-NtFD1 interaction in plant cytosol, leading to the degradation of NtFD1 in a ubiquitin-dependent manner. Silencing BtFTSP1 has negative effects on B. tabaci feeding while overexpressing BtFTSP1 in N. tabacum benefits insects and rescues the adverse effect caused by NtFD1 overexpression. The association between BtFTSP1 and NtFD1 is newly evolved after HGT, with the homologous FTSP in its fungal donor failing to interact and destabilize NtFD1. Our study illustrates the important roles of horizontally transferred genes in plant-insect interactions and suggests the potential origin of orphan salivary genes.