GPCR-MAPK signaling pathways underpin fitness trade-offs in whitefly.

Trade-offs between evolutionary gain and loss are prevalent in nature, yet their genetic basis is not well resolved. The evolution of insect resistance to insecticide is often associated with strong fitness costs; however, how the fitness trade-offs operates remains poorly understood. Here, we show that the mitogen-activated protein kinase (MAPK) pathway and its upstream and downstream actors underlie the fitness trade-offs associated with insecticide resistance in the whitefly Bemisia tabaci. Specifically, we find a key cytochrome P450 gene CYP6CM1, that confers neonicotinoids resistance to in B. tabaci, is regulated by the MAPKs p38 and ERK through their activation of the transcription factor cAMP-response element binding protein. However, phosphorylation of p38 and ERK also leads to the activation of the transcription repressor Cap "n" collar isoform C (CncC) that negatively regulates exuperantia (Ex), vasa (Va), and benign gonial cell neoplasm (Bg), key genes involved in oogenesis, leading to abnormal ovary growth and a reduction in female fecundity. We further demonstrate that the transmembrane G protein-coupled receptor (GPCR) neuropeptide FF receptor 2 (NPFF2) triggers the p38 and ERK pathways via phosphorylation. Additionally, a positive feedback loop between p38 and NPFF2 leads to the continuous activation of the MAPK pathways, thereby constitutively promoting neonicotinoids resistance but with a significant reproductive cost. Collectively, these findings provide fundamental insights into the role of cis-trans regulatory networks incurred by GPCR-MAPK signaling pathways in evolutionary trade-offs and applied knowledge that can inform the development of strategies for the sustainable pest control.

Knockdown of the salivary protein gene NlG14 caused displacement of the lateral oviduct secreted components and inhibited ovulation in Nilaparvata lugens.

Saliva plays important roles in insect feeding, but its roles in insect reproduction were rarely reported. Here we reported that the knockdown of a salivary gland-specific gene NlG14 disrupted the reproduction through inhibiting the ovulation of the brown planthopper (BPH), Nilaparvata lugens (Stål), one of the most devastating rice pests in Asia. NlG14 knockdown caused the displacement of the lateral oviduct secreted components (LOSC), leading to the ovulation disorder and the accumulation of mature eggs in the ovary. The RNAi-treated females laid much less eggs than their control counterparts, though they had the similar oviposition behavior on rice stems as controls. NlG14 protein was not secreted into the hemolymph, indicating an indirect effect of NlG14 knockdown on BPH reproduction. NlG14 knockdown caused the malformation of A-follicle of the principal gland and affected the underlying endocrine mechanism of salivary glands. NlG14 reduction might promote the secretion of insulin-like peptides NlILP1 and NlILP3 from the brain, which up-regulated the expression of Nllaminin gene and then caused the abnormal contraction of lateral oviduct muscle. Another explanation was NlG14 reduction disrupted the ecdysone biosynthesis and action through the insulin-PI3K-Akt signaling in ovary. Altogether, this study indicated that the salivary gland specific protein NlG14 indirectly mediated BPH ovulation process, which established a connexon in function between insect salivary gland and ovary.

Symbiotic bacteria confer insecticide resistance by metabolizing buprofezin in the brown planthopper, Nilaparvata lugens (Stål).

Buprofezin, a chitin synthesis inhibitor, is widely used to control several economically important insect crop pests. However, the overuse of buprofezin has led to the evolution of resistance and exposed off-target organisms present in agri-environments to this compound. As many as six different strains of bacteria isolated from these environments have been shown to degrade buprofezin. However, whether insects can acquire these buprofezin-degrading bacteria from soil and enhance their own resistance to buprofezin remains unknown. Here we show that field strains of the brown planthopper, Nilaparvata lugens, have acquired a symbiotic bacteria, occurring naturally in soil and water, that provides them with resistance to buprofezin. We isolated a symbiotic bacterium, Serratia marcescens (Bup_Serratia), from buprofezin-resistant N. lugens and showed it has the capacity to degrade buprofezin. Buprofezin-susceptible N. lugens inoculated with Bup_Serratia became resistant to buprofezin, while antibiotic-treated N. lugens became susceptible to this insecticide, confirming the important role of Bup_Serratia in resistance. Sequencing of the Bup_Serratia genome identified a suite of candidate genes involved in the degradation of buprofezin, that were upregulated upon exposure to buprofezin. Our findings demonstrate that S. marcescens, an opportunistic pathogen of humans, can metabolize the insecticide buprofezin and form a mutualistic relationship with N. lugens to enhance host resistance to buprofezin. These results provide new insight into the mechanisms underlying insecticide resistance and the interactions between bacteria, insects and insecticides in the environment. From an applied perspective they also have implications for the control of highly damaging crop pests.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Response of Citrus sinensis to Diaphorina citri Inoculation of Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) is a fatal citrus disease that is currently threatening citrus varieties worldwide. One putative causative agent, Candidatus Liberibacter asiaticus (CLas), is vectored by Diaphorina citri, known as the Asian citrus psyllid (ACP). Understanding the details of CLas infection in HLB disease has been hindered by its Candidatus nature and the inability to confidently detect it in diseased trees during the asymptomatic stage. To identify early changes in citrus metabolism in response to inoculation of CLas using its natural psyllid vector, leaves from Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees were exposed to CLas-positive ACP or CLas-negative ACP and longitudinally analyzed using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry; data available in Dryad: 10.25338/B83H1Z), and metabolomics (proton nuclear magnetic resonance). At 4 weeks postexposure (wpe) to psyllids, the initial HLB plant response was primarily to the ACP and, to a lesser extent, the presence or absence of CLas. Additionally, analysis of 4, 8, 12, and 16 wpe identified 17 genes and one protein as consistently differentially expressed between leaves exposed to CLas-positive ACP versus CLas-negative ACP. This study informs identification of early detection molecular targets and contributes to a broader understanding of vector-transmitted plant pathogen interactions.

The function of the cytoplasmic dynein light chain PTKM23 in the transport of PTSMAD2 during spermatogenesis in Portunus trituberculatus†.

Cytoplasmic dynein participates in transport functions and is essential in spermatogenesis. KM23 belongs to the dynein light chain family. The TGFß signaling pathway is indispensable in spermatogenesis, and Smad2 is an important member of this pathway. We cloned PTKM23 and PTSMAD2 from Portunus trituberculatus and measured their expression during spermatogenesis. PTKM23 may be related to cell division, acrosome formation, and nuclear remodeling, and PTSMAD2 may participate in regulating the expression of genes related to spermatogenesis. We assessed the localization of PTKM23 with PTDHC and α-tubulin, and the results suggested that PTKM23 functions in intracellular transport during spermatogenesis. We knocked down PTKM23 in vivo, and the expression of p53, B-CATAENIN and CYCLIN B decreased significantly, further suggesting a role of PTKM23 in transport and cell division. The localization of PTDIC with α-tubulin and that of PTSMAD2 with PTDHC changed after PTKM23 knockdown. We transfected PTKM23 and PTSMAD2 into HEK-293 T cells and verified their colocalization. These results indicate that PTKM23 is involved in the assembly of cytoplasmic dynein and microtubules during spermatogenesis and that PTKM23 mediates the participation of cytoplasmic dynein in the transport of PTSMAD2 during spermatogenesis.

Plasmodesmata-associated Flotillin positively regulates broad-spectrum virus cell-to-cell trafficking.

Viral diseases seriously threaten rice production. Plasmodesmata (PD)-associated proteins are deemed to play a key role in viral infection in host plants. However, few PD-associated proteins have been discovered in rice to afford viral infection. Here, inspired by the infection mechanism in insect vectors, we identified a member of the Flotillin family taking part in the cell-to-cell transport of rice stripe virus (RSV) in rice. Flotillin1 interacted with RSV nucleocapsid protein (NP) and was localized on PD. In flotillin1 knockout mutant rice, which displayed normal growth, RSV intercellular movement was retarded, leading to significantly decreased disease incidence. The PD pore sizes of the mutant rice were smaller than those of the wild type due to more callose deposits, which was closely related to the upregulation of two callose synthase genes. RSV infection stimulated flotillin1 expression and enlarged the PD aperture via RSV NP. In addition, flotillin1 knockout decreased disease incidences of southern rice black-streaked dwarf virus (SRBSDV) and rice dwarf virus (RDV) in rice. Overall, our study reveals a new PD-associated protein facilitating virus cell-to-cell trafficking and presents the potential of flotillin1 as a target to produce broad-spectrum antiviral rice resources in the future.

The essential role of clathrin-mediated endocytosis and early endosomes in the trafficking of begomoviruses through the primary salivary glands of their whitefly vectors.

IMPORTANCE: Many plant viruses are transmitted by insect vectors in a circulative manner. For efficient transmission, the entry of the virus from vector hemolymph into the primary salivary gland (PSG) is a step of paramount importance. Yet, vector components mediating virus entry into PSG remain barely characterized. Here, we demonstrate the role of clathrin-mediated endocytosis and early endosomes in begomovirus entry into whitefly PSG. Our findings unravel the key components involved in begomovirus transport within the whitefly body and transmission by their whitefly vectors and provide novel clues for blocking begomovirus transmission.

Interaction between endogenous microRNAs and virus-derived small RNAs controls viral replication in insect vectors.

MicroRNAs (miRNAs) play an important role in resisting virus infection in insects. Viruses are recognized by insect RNA interference systems, which generate virus-derived small RNAs (vsRNAs). To date, it is unclear whether viruses employ vsRNAs to regulate the expression of endogenous miRNAs. We previously found that miR-263a facilitated the proliferation of rice stripe virus (RSV) in the insect vector small brown planthopper. However, miR-263a was significantly downregulated by RSV. Here, we deciphered the regulatory mechanisms of RSV on miR-263a expression. The promoter region of miR-263a was characterized, and the transcription factor YY1 was found to negatively regulate the transcription of miR-263a. The nucleocapsid protein of RSV promoted the inhibitory effect of YY1 on miR-263a transcription by reducing the binding ability of RNA polymerase II to the promoter of miR-263a. Moreover, an RSV-derived small RNA, vsR-3397, downregulated miR-263a transcription by directly targeting the promoter region with partial sequence complementarity. The reduction in miR-263a suppressed RSV replication and was beneficial for maintaining a tolerable accumulation level of RSV in insect vectors. This dual regulation mechanism reflects an ingenious adaptation strategy of viruses to their insect vectors.

Phytochrome B mediates dim-light-reduced insect resistance by promoting the ethylene pathway in rice.

Increasing planting density is one of the most effective ways to improve crop yield. However, one major factor that limits crop planting density is the weakened immunity of plants to pathogens and insects caused by dim light (DL) under shade conditions. The molecular mechanism underlying how DL compromises plant immunity remains unclear. Here, we report that DL reduces rice (Oryza sativa) resistance against brown planthopper (BPH; Nilaparvata lugens) by elevating ethylene (ET) biosynthesis and signaling in a Phytochrome B (OsPHYB)-dependent manner. The DL-reduced BPH resistance is relieved in osphyB mutants, but aggravated in OsPHYB overexpressing plants. Further, we found that DL reduces the nuclear accumulation of OsphyB, thus alleviating Phytochrome Interacting Factor Like14 (OsPIL14) degradation, consequently leading to the up-regulation of 1-Aminocyclopropane-1-Carboxylate Oxidase1 (OsACO1) and an increase in ET levels. In addition, we found that nuclear OsphyB stabilizes Ethylene Insensitive Like2 (OsEIL2) by competitively interacting with EIN3 Binding F-Box Protein (OsEBF1) to enhance ET signaling in rice, which contrasts with previous findings that phyB blocks ET signaling by facilitating Ethylene Insensitive3 (EIN3) degradation in other plant species. Thus, enhanced ET biosynthesis and signaling reduces BPH resistance under DL conditions. Our findings provide insights into the molecular mechanism of the light-regulated ET pathway and host-insect interactions and potential strategies for sustainable insect management.

Silencing NlFAR7 destroyed the pore canals and related structures of the brown planthopper.

Fatty acyl-CoA reductase (FAR) is one of the key enzymes, which catalyses the conversion of fatty acyl-CoA to the corresponding alcohols. Among the FAR family members in the brown planthopper (Nilaparvata lugens), NlFAR7 plays a pivotal role in both the synthesis of cuticular hydrocarbons and the waterproofing of the cuticle. However, the precise mechanism by which NlFAR7 influences the formation of the cuticle structure in N. lugens remains unclear. Therefore, this paper aims to investigate the impact of NlFAR7 through RNA interference, transmission electron microscope, focused ion beam scanning electron microscopy (FIB-SEM) and lipidomics analysis. FIB-SEM is employed to reconstruct the three-dimensional (3D) architecture of the pore canals and related cuticle structures in N. lugens subjected to dsNlFAR7 and dsGFP treatments, enabling a comprehensive assessment of changes in the cuticle structures. The results reveal a reduction in the thickness of the cuticle and disruptions in the spiral structure of pore canals, accompanied by widened base and middle diameters. Furthermore, the lipidomics comparison analysis between dsNlFAR7- and dsGFP-treated N. lugens demonstrated that there were 25 metabolites involved in cuticular lipid layer synthesis, including 7 triacylglycerols (TGs), 5 phosphatidylcholines (PCs), 3 phosphatidylethanolamines (PEs) and 2 diacylglycerols (DGs) decreased, and 4 triacylglycerols (TGs) and 4 PEs increased. In conclusion, silencing NlFAR7 disrupts the synthesis of overall lipids and destroys the cuticular pore canals and related structures, thereby disrupting the secretion of cuticular lipids, thus affecting the cuticular waterproofing of N. lugens. These findings give significant attention with reference to further biochemical researches on the substrate specificity of FAR protein, and the molecular regulation mechanisms during N. lugens life cycle.

An association between Dnmt1 and Wnt in the production of oocytes in the whitefly Bemisia tabaci.

The function of DNA methylation in insects and the DNA methyltransferase (Dnmt) genes that influence methylation remains uncertain. We used RNA interference to reduce the gene expression of Dnmt1 within the whitefly Bemisia tabaci (Hemiptera:Aleyrodidae; Gennadius), a hemipteran species that relies on Dnmt1 for proper gametogenesis. We then used RNA-seq to test an a priori hypothesis that meiosis-related genetic pathways would be perturbed. We generally did not find an overall effect on meiosis-related pathways. However, we found that genes in the Wnt pathway, genes associated with the entry into meiosis in vertebrates, were differentially expressed. Our results are consistent with Dnmt1 knockdown influencing specific pathways and not causing general transcriptional response. This is a finding that is also seen with other insect species. We also characterised the methylome of B. tabaci and assessed the influence of Dnmt1 knockdown on cytosine methylation. This species has methylome characteristics comparable to other hemipterans regarding overall level, enrichment within gene bodies, and a bimodal distribution of methylated/non-methylated genes. Very little differential methylation was observed, and difference in methylation were not associated with differences in gene expression. The effect on Wnt presents an interesting new candidate pathway for future studies.

Constitutively expressed small heat shock protein LsHsp21.5 not only enhances heat tolerance but also helps to maintain reproduction in female Laodelphax striatellus.

Coping with stressful conditions and maintaining reproduction are two key biological processes that affect insect population dynamics. Small heat shock proteins (sHSPs) are involved in the stress response and the development of insects. The sHsp gene Laodelphax striatellus (Hemiptera: Delphacidae) sHsp 21.5 (LsHsp21.5) showed constitutive, stage- and organ-specific expression in L. striatellus, a pest that damages cultivated rice in east Asia. The expression of LsHsp21.5 was highest in the ovary, with 43.60, 12.99 and 1.45 time higher expression here than in the head, gut and female fat bodies, respectively. The expression of this gene was weakly affected by heat or cold shock. The gene provided in vitro protection against heat damage to malate dehydrogenase and in vivo protection against heat stress in Escherichia coli (Enterobacteriales: Enterobacteriaceae) BL21(DE3) and L. striatellus. Moreover, L. striatellus reproduction decreased by 1.85 times when the expression of LsHsp21.5 was inhibited by RNA interference. The expression of some genes related to reproduction, such as the homologous gene of chorion protein, also declined. These results suggest that LsHsp21.5 expression not only protects other proteins against stress but also helps maintain the stable expression of some reproduction-related genes under non-stressful conditions, with impacts on L. striatellus fecundity.

The growth factor BMP11 is required for the development and evolution of a male exaggerated weapon and its associated fighting behavior in a water strider.

Exaggerated sexually selected traits, often carried by males, are characterized by the evolution of hyperallometry, resulting in their disproportionate growth relative to the rest of the body among individuals of the same population. While the evolution of allometry has attracted much attention for centuries, our understanding of the developmental genetic mechanisms underlying its emergence remains fragmented. Here we conduct comparative transcriptomics of the legs followed by an RNA interference (RNAi) screen to identify genes that play a role in the hyperallometric growth of the third legs in the males of the water strider Microvelia longipes. We demonstrate that a broadly expressed growth factor, Bone Morphogenetic Protein 11 (BMP11, also known as Growth Differentiation Factor 11), regulates leg allometries through increasing the allometric slope and mean body size in males. In contrast, BMP11 RNAi reduced mean body size but did not affect slope either in the females of M. longipes or in the males and females of other closely related Microvelia species. Furthermore, our data show that a tissue-specific factor, Ultrabithorax (Ubx), increases intercept without affecting mean body size. This indicates a genetic correlation between mean body size and variation in allometric slope, but not intercept. Strikingly, males treated with BMP11 RNAi exhibited a severe reduction in fighting frequency compared to both controls and Ubx RNAi-treated males. Therefore, male body size, the exaggerated weapon, and the intense fighting behavior associated with it are genetically correlated in M. longipes. Our results support a possible role of pleiotropy in the evolution of allometric slope.

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.

Vestigial mediates the effect of insulin signaling pathway on wing-morph switching in planthoppers.

Wing polymorphism is an evolutionary feature found in a wide variety of insects, which offers a model system for studying the evolutionary significance of dispersal. In the wing-dimorphic planthopper Nilaparvata lugens, the insulin/insulin-like growth factor signaling (IIS) pathway acts as a 'master signal' that directs the development of either long-winged (LW) or short-winged (SW) morphs via regulation of the activity of Forkhead transcription factor subgroup O (NlFoxO). However, downstream effectors of the IIS-FoxO signaling cascade that mediate alternative wing morphs are unclear. Here we found that vestigial (Nlvg), a key wing-patterning gene, is selectively and temporally regulated by the IIS-FoxO signaling cascade during the wing-morph decision stage (fifth-instar stage). RNA interference (RNAi)-mediated silencing of Nlfoxo increase Nlvg expression in the fifth-instar stage (the last nymphal stage), thereby inducing LW development. Conversely, silencing of Nlvg can antagonize the effects of IIS activity on LW development, redirecting wing commitment from LW to the morph with intermediate wing size. In vitro and in vivo binding assays indicated that NlFoxO protein may suppress Nlvg expression by directly binding to the first intron region of the Nlvg locus. Our findings provide a first glimpse of the link connecting the IIS pathway to the wing-patterning network on the developmental plasticity of wings in insects, and help us understanding how phenotypic diversity is generated by the modification of a common set of pattern elements.

Neofunctionalization of a second insulin receptor gene in the wing-dimorphic planthopper, Nilaparvata lugens.

A single insulin receptor (InR) gene has been identified and extensively studied in model species ranging from nematodes to mice. However, most insects possess additional copies of InR, yet the functional significance, if any, of alternate InRs is unknown. Here, we used the wing-dimorphic brown planthopper (BPH) as a model system to query the role of a second InR copy in insects. NlInR2 resembled the BPH InR homologue (NlInR1) in terms of nymph development and reproduction, but revealed distinct regulatory roles in fuel metabolism, lifespan, and starvation tolerance. Unlike a lethal phenotype derived from NlInR1 null, homozygous NlInR2 null mutants were viable and accelerated DNA replication and cell proliferation in wing cells, thus redirecting short-winged-destined BPHs to develop into long-winged morphs. Additionally, the proper expression of NlInR2 was needed to maintain symmetric vein patterning in wings. Our findings provide the first direct evidence for the regulatory complexity of the two InR paralogues in insects, implying the functionally independent evolution of multiple InRs in invertebrates.

Influence of Cry1Ab protein on growth and development of a predatory spider, Pardosa pseudoannulata, from protective perspectives.

The expression of Cry proteins in genetically modified rice varieties safeguards the crop from lepidopteran pests. These proteins have the potential to be transferred through the food chain to arthropods like planthoppers and predatory spiders, triggering defensive responses in these unintended organisms. Hence, we hypothesized that Cry protein might influence the growth and development of spiders by altering protective enzyme activities. The results showed that Cry1Ab protein could accumulate in tissues and subcellular organelles of Pardosa pseudoannulata from Nilaparvata lugens. Cry1Ab protein exposure prolonged the developmental duration in the 5th and 7th instar spiderlings but induced no alterations of other growth indicators, such as body length, median ocular area, and survival rate. In addition, Cry1Ab protein exerted no adverse impacts on several detoxifying enzymes (i.e., superoxide dismutase, catalase, glutathione peroxidase, and acetylcholine esterase) in muscle, midgut, ganglia, and hemolymph at subcellular components (i.e., microsome and cytoplasm). To further explore the effects of Cry1Ab protein on the spiderlings, we performed an integrated transcriptome analysis on spiderlings exposed to Cry1Ab protein. The results showed that Cry1Ab protein might prolong the development duration of P. pseudoannulata via the altered cuticle metabolism (e.g., chitin metabolic process and structural constituent of cuticle). In addition, the gene expression profile associated with detoxifying enzymes and three stress-responsive pathways (JAK/STAT, JNK/SAPK, and Hippo pathways) also displayed no significant alterations under Cry1Ab exposure. Collectively, this integrated analysis generates multidimensional insights to assess the effects of Cry1Ab protein on non-target spiders and demonstrates that Cry1Ab protein exerts no toxicity in P. pseudoannulata.

RpedOBP1 plays key roles in aggregation pheromones reception of the Riptortus pedestris.

Riptortus pedestris (Hemiptera: Alydidae) is a notable soybean pest, with diapause and non-diapause individuals showing different sensitivities to aggregation pheromones. This study aimed to investigate how R. pedestris detects aggregation pheromones through electroantennogram (EAG) and behavioral experiments, transcriptome sequencing and qRT-PCR, as well as competitive fluorescence-binding assay. Results indicated that diapausing females and males of R. pedestris exhibited a heightened EAG response and were more attracted to the aggregation pheromone components compared to their non-diapause counterparts. Transcriptome sequencing and qRT-PCR analyses revealed significantly higher expression of RpedOBP1 in the antennae of diapause females and males compared to non-diapausing R. pedestris. The competitive fluorescence-binding assay demonstrated that RpedOBP1 displayed the strongest binding affinity to E2HE2H, suggesting its crucial role in recognizing the aggregation pheromone. These findings have the potential to inform the development of integrated pest management strategies utilizing behavioral approaches for bean bug control.

Chemosensory protein 22 in Riptortus pedestris is involved in the recognition of three soybean volatiles.

Riptortus pedestris (Hemiptera: Alydidae), a common agricultural pest, is the major causative agent of "soybean staygreen." However, the interactions between chemosensory proteins (CSPs) in R. pedestris and host plant volatiles have yet to be comprehensively studied. In this study, we performed real-time fluorescence quantitative polymerase chain reaction (PCR) to analyze the antennal expression of RpedCSP22 and subsequently analyzed the interactions between 21 soybean volatiles, five aggregation pheromones, and RpedCSP22 protein in vitro using a protein expression system, molecular docking, site-directed mutagenesis, and fluorescence competitive binding experiments. The RpedCSP22 protein showed binding affinity to three soybean volatiles (benzaldehyde, 4-ethylbenzaldehyde, and 1-octene-3-ol), with optimal binding observed under neutral pH conditions, and lost binding ability after site-directed mutagenesis. In subsequent RNA interference (RNAi) studies, gene silencing was more than 90 %, and in silenced insects, electroantennographic responses were reduced by more than 75 % compared to non-silenced insects. Moreover, Y-tube olfactory behavioral assessments revealed that the attraction of R. pedestris to the three soybean volatiles was significantly attenuated. These findings suggest that RpedCSP22 plays an important role in the recognition of host plant volatiles by R. pedestris andprovides a theoretical basis for the development of novel inhibitors targeting pest behavior.

Effectiveness of chitosan nanohydrogel mediated encapsulation of EcR dsRNA against the whitefly, Bemisia tabaci Asia-I (Gennedius) (Hemiptera: Aleyordidae).

Bemisia tabaci is a global invasive pest causing substantial loss on several economically important crops and has developed a very high level of resistance to insecticides making current management practices ineffective. Thus, the novel pest management strategy like RNA interference (RNAi) has emerged as a potential molecular tool in the management of insect pests particularly B. tabaci. The present study investigated RNAi mediated silencing of the Ecdysone Receptor (EcR) gene in B. tabaci Asia-I using biodegradable Chitosan Nanoparticles (CNPs) hydrogel containing EcR dsRNA. The formation of nanohydrogel and dsRNA loading were characterized by gel retardation assay, scanning electron microscopy (SEM); transmission electron microscopy (TEM) and Fourier transform infrared microscopy (FTIR). The stability of CNPs/dsRNA was assessed by exposure to direct sunlight and UV light for different time periods. The CNPs/dsRNA exhibited increased stability over the untreated control and further confirmed by bioassay studies which yielded mortality over 80% and effectively down regulated the expression of the EcR gene as confirmed by qRT-PCR analysis. These investigations provide potential avenues for advancing innovative pest management strategies using biopolymer CNPs hydrogel, which can enhance the efficiency of dsRNA as a safe and targeted solution in the management of whiteflies.