Electron Tomography and Machine Learning for Understanding the Highly Ordered Structure of Leafhopper Brochosomes.

Insects known as leafhoppers (Hemiptera: Cicadellidae) produce hierarchically structured nanoparticles known as brochosomes that are exuded and applied to the insect cuticle, thereby providing camouflage and anti-wetting properties to aid insect survival. Although the physical properties of brochosomes are thought to depend on the leafhopper species, the structure-function relationships governing brochosome behavior are not fully understood. Brochosomes have complex hierarchical structures and morphological heterogeneity across species, due to which a multimodal characterization approach is required to effectively elucidate their nanoscale structure and properties. In this work, we study the structural and mechanical properties of brochosomes using a combination of atomic force microscopy (AFM), electron microscopy (EM), electron tomography, and machine learning (ML)-based quantification of large and complex scanning electron microscopy (SEM) image data sets. This suite of techniques allows for the characterization of internal and external brochosome structures, and ML-based image analysis methods of large data sets reveal correlations in the structure across several leafhopper species. Our results show that brochosomes are relatively rigid hollow spheres with characteristic dimensions and morphologies that depend on leafhopper species. Nanomechanical mapping AFM is used to determine a characteristic compression modulus for brochosomes on the order of 1-3 GPa, which is consistent with crystalline proteins. Overall, this work provides an improved understanding of the structural and mechanical properties of leafhopper brochosomes using a new set of ML-based image classification tools that can be broadly applied to nanostructured biological materials.

Sex Pheromone of the Azalea Mealybug With a Non-Terpene Structure.

Mealybug females release sex pheromones to attract conspecific males for mating. It is critical for mealybug males, which are fragile and short-lived, to respond to the pheromone of their species without time- and energy-consuming cross-attractions to other species. Thus, mealybug pheromone systems are considered to have evolved to be species-specific with unique structures in each species and offer an opportunity to study the diversity of pheromone chemistry that mediates intersexual courtship signals. More than 20 mealybug pheromones are reported to be monoterpenes in general, with only one exception, a hemiterpene alcohol esterified with a medium-chain fatty acid (MCFA), found in the Matsumoto mealybug, Crisicoccus matsumotoi. However, it is unknown whether this is truly exceptional, or if similar compounds are used in other related mealybugs. In this study, we isolated and characterized the pheromone of an allied species, the azalea mealybug C. azaleae. Using gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and bioassays with synthetics, the pheromone was shown to be composed of isopropyl (E)-7-methyl-4-nonenoate, isopropyl (E)-7-methyl-4-octenoate, and ethyl (E)-7-methyl-4-nonenoate. Surprisingly, the structures of these compounds do not include hemiterpene nor monoterpene motifs but have methyl-branched MCFA parts that are similar to an acid moiety of the C. matsumotoi pheromone. This study implies irregular events for the divergence of pheromone structures in ancestors of the genus Crisicoccus and other mealybugs.

Ovary structure and symbiotic associates of a ground mealybug, Rhizoecus albidus (Hemiptera, Coccomorpha: Rhizoecidae) and their phylogenetic implications.

The ovary structure and the organization of its symbiotic system of the ground mealybug, Rhizoecus albidus (Rhizoecidae), were examined by means of microscopic and molecular methods. Each of the paired elongated ovaries of R. albidus is composed of circa one hundred short telotrophic-meroistic ovarioles, which are radially arranged along the distal part of the lateral oviduct. Analysis of serial sections revealed that each ovariole contains four germ cells: three trophocytes (nurse cells) occupying the tropharium and a single oocyte in the vitellarium. The ovaries are accompanied by giant cells termed bacteriocytes which are tightly packed with large pleomorphic bacteria. Their identity as Brownia rhizoecola (Bacteroidetes) was confirmed by means of amplicon sequencing and fluorescence in situ hybridization techniques. Moreover, to our knowledge, this is the first report on the morphology and ultrastructure of the Brownia rhizoecola bacterium. In the bacteriocyte cytoplasm bacteria Brownia co-reside with sporadic rod-shaped smaller bacteria, namely Wolbachia (Proteobacteria: Alphaproteobacteria). Both symbionts are transmitted to the next generation vertically (maternally), that is, via female germline cells. We documented that, at the time when ovarioles contain oocytes at the vitellogenic stage, these symbionts leave the bacteriocytes and move toward the neck region of ovarioles (i.e. the region between tropharium and vitellarium). Next, the bacteria enter the cytoplasm of follicular cells surrounding the basal part of the tropharium, leave them and enter the space between the follicular epithelium and surface of the nutritive cord connecting the tropharium and vitellarium. Finally, they gather in the deep depression of the oolemma at the anterior pole of the oocyte in the form of a 'symbiont ball'. Our results provide further arguments strongly supporting the validity of the recent changes in the classification of mealybugs, which involved excluding ground mealybugs from the Pseudococcidae family and raising them to the rank of their own family Rhizoecidae.

Cicada-Wing-Inspired Nanopillar Hydrogels Consisting of Poly(vinyl alcohol) and Poly(methacrylic acid) for Capturing Bacteria through Their Flexibility and Wide Range of Motion.

The human race has learned about countering bacterial infection from other living organisms. In this study, cicada-wing-inspired hydrophilic nanopillars were prepared using commercially available porous alumina templates without the use of specialized equipment and additional materials. Hydrophilic polymers of poly(vinyl alcohol) (PVA) and poly(methacrylic acid) (poly(MAAc)) were used to construct these nanopillars. The water-insoluble nanopillars formed by thermal cross-linking between the hydroxy and carboxy groups were highly flexible, with no significant structural disruption being observed on tilting them. The nanopillars captured Gram-negative and Gram-positive bacteria, despite their negatively charged surface leading an electrostatic repulsion with bacteria. The wing of the cicada has evolved to remain dry and maintain its lightweight. The water-containing cicada-wing structure may be an unknown encounter for bacteria and, thus, may become a countermeasure against the ever-changing bacteria.

Genetic Basis Underlying Structural Shift of Monoterpenoid Pheromones in Mealybugs.

Insect sex pheromones are examples of semiochemicals that trigger the most conspicuous biological activities, and they have attracted the interest of chemical ecologists since the dawn of this multidisciplinary field. For a deeper understanding of the ecological and evolutionary scenario of pheromones, as well as other targets of chemical ecology, it is essential to analyze the chemicals produced by individual organisms along with sound chemical identifications using reference compounds. Prof. Kenji Mori and his colleagues have developed various synthetic routes and have provided their products as authentic standards to many researchers. Using such a legacy, the tiny amounts of pheromones emitted by individual mealybug females were successfully analyzed and quantified by selected-ion-monitoring mode of gas chromatography-mass spectrometry. The results of the analyses of the monoterpene pheromones from Planococcus citri, P. minor, and their hybrids suggested that shift of the cyclobutane structure in P. citri and its acyclic form in P. minor is largely attributable to a single genetic locus.

Phototoxicity of Ultraviolet-A against the Whitefly Bemisia tabaci and Its Compatibility with an Entomopathogenic Fungus and Whitefly Parasitoid.

Ultraviolet (UV) radiation significantly affects insect life and, as a result, has been widely used to control different invertebrate pests. The current results demonstrate that when Bemisia tabaci first instar nymphs are exposed to UV-A light for 12, 24, 48, and 72 h, their developmental and biological parameters are negatively affected by UV-A exposure; the effect increased with an increase in exposure time. We hypothesized that UV-A light is compatible with other biological control agents. Results showed that when the entomopathogenic fungus Cordyceps fumosorosea was applied to third instar nymphs of B. tabaci previously exposed to UV-A light, the LC50 was 3.4% lower after 72 h of exposure to UV-A light compared to the control. However, when the fungus was exposed to UV-A light, its virulence decreased with an increase in UV-A exposure time. The parasitism rate of Encarsia formosa against 24 h UV-A-exposed third instar nymphs of B. tabaci increased while the adult emergence from parasitized nymphs was not affected after UV-A light exposure. Parasitism rate was significantly reduced however following E. formosa exposure to UV-A light; but again, adult emergence was not affected from parasitized nymphs. The percentage mortality of E. formosa increased with increasing exposure time to UV-A light. The enzyme activity of SOD, CAT, GST, and AChE and the energy reserve contents were negatively affected due to UV-A exposure. Collectively, this study has demonstrated that UV-A light significantly suppresses the immune system of B. tabaci and that UV-A light is compatible with other biological control agents if it is applied separately from the biological agent.

Cryptotympana pustulata Extract and Its Main Active Component, Oleic Acid, Inhibit Ovalbumin-Induced Allergic Airway Inflammation through Inhibition of Th2/GATA-3 and Interleukin-17/RORγt Signaling Pathways in Asthmatic Mice.

Cicadae Periostracum (CP), derived from the slough of Cryptotympana pustulata, has been used as traditional medicine in Korea and China because of its diaphoretic, antipyretic, anti-inflammatory, antioxidant, and antianaphylactic activities. The major bioactive compounds include oleic acid (OA), palmitic acid, and linoleic acid. However, the precise therapeutic mechanisms underlying its action in asthma remain unclear. The objective of this study was to determine the antiasthmatic effects of CP in an ovalbumin (OVA)-induced asthmatic mouse model. CP and OA inhibited the inflammatory cell infiltration, airway hyperresponsiveness (AHR), and production of interleukin (IL)7 and Th2 cytokines (IL-5) in the bronchoalveolar lavage fluid and OVA-specific imunoglobin E (IgE) in the serum. The gene expression of IL-5, IL-13, CCR3, MUC5AC, and COX-2 was attenuated in lung tissues. CP and OA might inhibit the nuclear translocation of GATA-binding protein 3 (GATA-3) and retinoic acid receptor-related orphan receptor γt (RORγt) via the upregulation of forkhead box p3 (Foxp3), thereby preventing the activation of GATA-3 and RORγt. In the in vitro experiment, a similar result was observed for Th2 and GATA-3. These results suggest that CP has the potential for the treatment of asthma via the inhibition of the GATA-3/Th2 and IL-17/RORγt signaling pathways.

Estimating included animal species in mixed crude drugs derived from animals using massively parallel sequencing.

We developed a method that can detect each animal species of origin for crude drugs derived from multiple animal species based on massively parallel sequencing analysis of mitochondrial genes. The crude drugs derived from animals investigated in this study were Cervi Parvum Cornu and Trogopterorum feces, which are derived from a mix of different animal species, two chopped cicada sloughs, and two commercial Kampo drugs. The mitochondrial 12S rRNA, 16S rRNA, and cytochrome oxidase subunit I gene regions were amplified and sequenced using MiSeq. The ratios of haplotype to total number of sequences reads were calculated after sequence extraction and trimming. Haplotypes that exceeded the threshold were defined as positive haplotypes, which were compared with all available sequences using BLAST. In the Cervi Parvum Cornu and Trogopterorum feces samples, the haplotype ratios corresponded roughly to the mixture ratios, although there was a slight difference from mixture ratios depending on the gene examined. This method could also roughly estimate the compositions of chopped cicada sloughs and Kampo drugs. This analysis, whereby the sequences of several genes are elucidated, is better for identifying the included animal species. This method should be useful for quality control of crude drugs and Kampo drugs.

Cicadidae Periostracum Attenuates Atopic Dermatitis Symptoms and Pathology via the Regulation of NLRP3 Inflammasome Activation.

Atopic dermatitis (AD) is a multifactorial inflammatory skin disease of complex etiology. Despite its increasing prevalence, treatment for AD is still limited. Crude drugs, including herbal extracts or natural resources, are being used to treat AD symptoms, with minimum side effects. Cicadidae Periostracum (CP), derived from the slough of insects belonging to the family Cicadidae, is a commonly used crude drug in traditional Asian medicine to treat/control epilepsy, shock, and edema. However, the effect of CP on AD-like skin lesions is unknown. In this study, we examined the effect of a CP water extract on AD disease development in vivo, using a house dust mite-induced AD mouse model, and in vitro, using HaCaT keratinocytes and a 3D human skin equivalent system. Importantly, CP administration alleviated house dust mite-induced AD-like symptoms, suggested by the quantified dermatitis scores, animal scratching behaviors, skin moisture retention capacity, and skin lesion and ear thickness. Furthermore, histopathological analysis demonstrated that CP decreased intralesional mast cell infiltration. In addition, CP treatments decreased the systemic levels of immunoglobulin E, histamine, and thymic stromal lymphopoietin (TSLP) and the local mRNA expression of TSLP and several Th1/Th2 cytokines. Our data suggest that these effects were mediated by the inhibition of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation. In vivo and in vitro CP treatments resulted in the downregulation of inflammasome components, such as ASC and cleaved caspase-1, as well as related mediators such as IL-1ß and reactive oxygen species. Collectively, our results suggest that CP is a potential therapeutic agent for AD, controlling inflammatory responses through the suppression of NLRP3 inflammasome activation.

Anti-androgenetic alopecia effect of policosanol from Chinese wax by regulating abnormal hormone levels to suppress premature hair follicle entry into the regression phase.

Previously, we have demonstrated that policosanol from Chinese wax suppressed testosterone(T)-induced alopecia in mice. However, the underlying mechanism remained to be determined. Herein, we investigated the mechanism of policosanol against androgenetic alopecia (AGA). AGA was induced in Kunming mice by subcutaneous administration of testosterone propionate for 60 d. Policosanol (0.5 %, 1% or 2%) was applied topically on the back of mice. Finasteride (2%) was applied topically as a positive control. The serum T and estradiol (E2) concentrations were determined by ELISA after 28 and 60 days of treatment. The cutaneous expression or activity of key mediators of hair growth, such as alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF), was measured. MTS assay was performed to evaluate cell proliferation in cultured human dermal papilla cells (DPCs) treated with dihydrotestosterone (DHT). Western blotting was performed to evaluate the protein expression of Bax, Bcl2, TGF-ß2, caspase-9, and caspase-3. We found lower T and T/E2 ratio in mice treated with policosanol than in the model group. Policosanol suppressed premature hair follicle entry into the regression phase, as shown by improving VEGF and EGF expression and ALP activity. The MTS assay showed that policosanol markedly inhibited the apoptosis of DHT-treated DPCs. Western blotting showed that policosanol significantly reduced the protein expression of TGF-ß2, cleaved caspese-9, cleaved caspase-3, and Bax, and increased that of Bcl2. The optimal effect was obtained with 12.50 g/mL policosanol. In conclusion, policosanol prevents androgenetic alopecia by regulating hormone levels and suppressing premature hair follicle entry into the regression phase.

Suntamide A, a neuroprotective cyclic peptide from Cicadidae Periostracum.

Suntamide A (1), a new cyclic peptide, was isolated from Cicadidae Periostracum. The gross structure of 1 was elucidated by detailed analysis of HRMS and 1D/2D NMR spectra, and the absolute configuration was established by C3 Marfey's method. We extended our study to examine biological activity of 1, and found that 1 protected SH-SY5Y cells against rotenone-induced neurotoxicity. This effect of 1 seemed to be attributed to antioxidant induction and protection of mitochondria from rotenone-caused injury. Along with augmentation of the antioxidant system by 1, there was an evident activation of Nrf2, a transcription factor involved in the activation of the antioxidant system. These results indicate that 1 rescued the cells from rotenone-mediated neurotoxicity by enhancing antioxidant capacity via induction of Nrf2, suggesting that the compound could be used as a therapeutic intervention in neurodegenerative diseases such as Parkinson's disease.

Molecular characterization of N-methyl-d-aspartate receptor from Bemisia tabaci.

The N-methyl-d-aspartate receptors (NMDARs) are ionotropic ligand gated channels that are highly permeable to calcium ions. In insects, NMDARs are associated with glutamatergic neurotransmission governing diverse physiological and biological processes like vitellogenesis and ovarian development. Therefore, NMDAR may act as attractive target for insect pest control. In present study, we performed structural and functional characterization of NMDARs in Bemisia tabaci, a highly invasive crop pest and potent virus vector. We identified that NMDAR consists of three subunits each encoded by single gene in whiteflies which are highly conserved among different insect orders. Expression analysis suggests that subunit 1 (BtNR1) and subunit 2 (BtNR2) are the main functional units. External supplementation of NMDAR ligand or BtNRs silencing was lethal to insects, which suggested that NMDAR function is highly balanced in whiteflies.

Replica molding of cicada wings: The role of water at point of synthesis on nanostructure feature size.

Many natural surfaces, including the wings of cicada insects, have shown to display bactericidal properties as a result of surface topography. Moreover, the size and distribution of the surface features (on the nano- and microscale) are known to influence the efficacy of the surface at inhibiting bacterial cell growth. While these types of natural surfaces illustrate the effect of structure on the bactericidal activity, a deeper understanding can be achieved by creating surfaces of different feature sizes. This is essential in order to understand the effects of changes of surface topography on bacteria-surface interactions. To this end, we have performed a series of replica molding processes of the wings of the Megapomponia Intermedia cicada to prepare wing replicas in polyethylene glycol (PEG), which possess the topographical features of the wing surface, with a minimum loss of feature resolution. Atomic force microscopy characterization of these patterned surfaces in both air and aqueous environments shows that by controlling the swelling characteristics of the PEG, we can control the ultimate swollen dimensions of the nanopillar structures on the surface of PEG. As a result, by using a single wing with an average nanopillar height of 220 nm, different patterned PEG samples with nanopillar heights ranging from 180 to 307 nm were produced.

Adipokinetic hormone regulates cytochrome P450-mediated imidacloprid resistance in the brown planthopper, Nilaparvata lugens.

Insect resistance to chemical insecticide is a global problem that presents an ongoing threat to sustainable agriculture. Although the increased production of detoxification enzymes has been frequently implicated in resistance development, the mechanisms employed by insecticide-resistant insects for overexpression of these genes remain elusive. Here we report that neuropeptide adipokinetic hormone (AKH) negatively regulates the expression of CYP6ER1 and CYP6AY1, two important cytochrome P450 monooxygenases (P450s) that confer resistance to neonicotinoid imidacloprid in the brown planthopper (BPH). Imidacloprid exposure suppresses AKH synthesis in the susceptible BPH, and AKH is inhibited in the imidacloprid-resistant strain. RNA interference (RNAi) and AKH peptide injection revealed that imidacloprid exposure inhibits the AKH signaling cascade and then provokes reactive oxygen species (ROS) burst. These in turn activate the transcription factors cap 'n' collar isoform-C (CncC) and muscle aponeurosis fibromatosis (MafK). RNAi and ROS scavenger assays showed that ROS induces CYP6ER1 expression by activating CncC and MafK, while ROS mediates induction of CYP6AY1 through another unidentified pathway in the resistant BPH. Collectively, these results provide new insights into the regulation of insecticide resistance and implicate both the neuropeptide AKH-mediated ROS burst and transcription factors are involved in the overexpression of P450 detoxification genes in insecticide-resistant insects.

N-Acetyldopamine derivatives from Periostracum Cicadae and their regulatory activities on Th1 and Th17 cell differentiation.

Bioassay-guided fractionation of a 90% ethanol extract of Periostracum Cicadae led to the isolation of two new N-acetyldopamine dimers (1a/1b) along with six known dimers (2a/2b, 3a/3b, and 4a/4b) and two monomers (5a/5b); compounds 2a/2b, 4a/4b and 5a/5b were newly isolated from this material. All compounds were isolated as enantiomeric mixtures and each enantiomer was successfully separated by chiral-phase HPLC. The structures including absolute configurations were confirmed by high-resolution electrospray ionization mass spectrometry (HR-ESIMS), 1D/2D nuclear magnetic resonance (NMR) spectroscopy, 1H iterative Full Spin Analysis (HiFSA), and electronic circular dichroism (ECD) spectroscopy. Subsequently, the bioactivities of these isolates were evaluated via CD4+ T cell differentiations, which are critical for immune responses and inflammation. The results revealed that compound 5b was observed to enhance the IFN-γ+ Th1 differentiation, which may have a potential for cancer immunotherapy.

In vitro redox activity of haemozoin and ß-haemozoin interacting with the following antimalarials: artemether, lumefantrine and quinine.

OBJECTIVE: Malaria parasites invade, grow and multiply inside erythrocytes and obtain nourishment from haemoglobin. Then, the released haem group is oxidized to haematin and inert dimeric haemozoin bio-crystals form, which provides the parasite a unique way to avoid the toxicity associated with the haem group. Therefore, antimalarial drugs are designed to inhibit dimer formation; however, recent electrochemical studies indicate that an inert dimer also promotes a toxic oxidizing environment. Therefore, this work explores drug reactivity in the presence of monomers and dimers to evaluate their contribution to redox activity. MATERIALS AND METHODS: Three medicines mixed with haemozoin or ß-haemozoin in carbon paste electrodes were tested using cyclic voltammetry. RESULTS: The data indicated again that the substances modify the natural redox state of haemozoin and ß-haemozoin. This effect could be attributed to the natural oxidation potential of the drugs. In addition, it was found that the oxidation potential decreased through quinine, lumefantrine and artemether with the same tendency in the presence of haemozoin but with less current density. Additionally, it was observed that the oxidation response between the monomer haemozoin and antimalarial drugs is carried out at more negative potentials. CONCLUSIONS: Together, the total results indicate that antimalarials per se can contribute to oxidation processes and that in combination with monomeric or dimeric haemozoin can increase or decrease the oxidizing power of the haemozoin forms. The various oxidizing environments suggest that the cell membranes can also be damaged by the unique presence of the antimalarial.

Extraction and Physicochemical Characterization of Chitin from Cicada orni Sloughs of the South-Eastern French Mediterranean Basin.

Chitin is a structural polysaccharide of the cell walls of fungi and exoskeletons of insects and crustaceans. In this study, chitin was extracted, for the first time in our knowledge, from the Cicada orni sloughs of the south-eastern French Mediterranean basin by treatment with 1 M HCl for demineralization, 1 M NaOH for deproteinization, and 1% NaClO for decolorization. The different steps of extraction were investigated by Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD), Thermogravimetric Analysis (TGA), and Scanning Electron Microscopy (SEM). Results demonstrated that the extraction process was efficiently performed and that Cicada orni sloughs of the south-eastern French Mediterranean basin have a high content of chitin (42.8%) in the α-form with a high degree of acetylation of 96% ± 3.4%. These results make Cicada orni of the south-eastern French Mediterranean basin a new and promising source of chitin. Furthermore, we showed that each step of the extraction present specific characteristics (for example FTIR and XRD spectra and, consequently, distinct absorbance peaks and values of crystallinity as well as defined values of maximum degradation temperatures identifiable by TGA analysis) that could be used to verify the effectiveness of the treatments, and could be favorably compared with other natural chitin sources.

The characteristic smell emitted from two scale insects, Ceroplastes japonicus and Ceroplastes rubens.

The volatile components emitted from two scale insects, Ceroplastes japonicus and Ceroplastes rubens, were identified using GC-MS analysis. The major volatile components of the solvent extract from C. japonicus were α-humulene (35.8%) and δ-cadinene (17.0%), while those of C. rubens were ß-selinene (10.3%) and ß-elemene (5.1%). In GC/olfactometry, linalool, butyric acid, 3-methylbutyric acid, 2-methylbutyric acid, and vanillin were identified as the odor-active components of the extract from C. japonicus, in addition to trace amounts of trans-4,5-epoxy-(2E)-decenal, 4-methyl-(3E)-hexenoic acid, and phenylacetic acid. With regard to C. rubens, trans-4,5-epoxy-(2E)-decenal, 3-methylbutyric acid, and phenylacetic acid were identified as the odor-active components. Besides, decan-1,4-olide (γ-decalactone) with milky cherry-like note and 3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolone) with brown sugar-like note were also detected as the characteristic cherry-like sweet-and-sour note of these two scale insects. ABBREVIATIONS: GC: Gas chromatography; GC/O: gas chromatography/olfactometry.

Safety assessment of transgenic Cry2Aa rice to a generalist predator, Paederus fuscipes Curtis (Coleoptera: Staphylinidae).

The insecticidal crystal proteins of Cry2A family from Bacillus thuringiensis (Bt) are important candidate proteins expressed in gene pyramiding Bt crops. A transgenic rice line (T2A-1) harboring a synthetic Cry2A* (Cry2Aa) gene showed effective resistance to some lepidopteran rice pests. As a generalist predator in rice ecosystems, the rove beetle (Paederus fuscipes) can prey on many rice insect pests such as planthoppers. Considering the possible exposure of Cry2Aa to P. fuscipes through tritrophic food chain, it is necessary to assess the potential risks of T2A-1 rice to this predator. In this study, a tritrophic experiment was conducted to assess the prey-mediated effects of Cry2Aa on P. fuscipes through the T2A-1 rice-Nilaparvata lugens-P. fuscipes food chain. After preying on N. lugens nymphs reared on T2A-1, no accumulated Cry2Aa could be detected in P. fuscipes adults, despite Cry2Aa being detected in N. lugens. In addition, no harmful effects were detected on the life table parameters of P. fuscipes in this tritrophic chain. Additionally, direct exposure to a high dose of purified Cry2Aa protein, representing the worst case scenario, showed no significant adverse effects on the development of P. fuscipes. These results showed that transgenic Cry2Aa rice had no harmful effects on P. fuscipes.

Expression Profiles and Biochemical Analysis of Chemosensory Protein 3 from Nilaparvata lugens (Hemiptera: Delphacidae).

Insects have evolved highly sensitive olfactory sensory systems to detect plant hosts and mates, with plant volatiles playing an important role in informing insect behavior. Chemosensory proteins (CSPs) are thought to play a key role in this process, but in this respect, there is limited information on brown planthopper Nilaparvata lugens, one of the most destructive pests of rice. To expand our understanding of CSP function in N. lugens we explored expression profiles and binding characteristics of NlugCSP3. The ligands with higher binding affinity were also validated by molecular docking and behavioral assays. NlugCSP3 mRNA was expressed at relatively higher levels in antennae and abdomen of 3-day-old unmated macropterous males as well as in antennae of 3-day mated macropterous and brachypterous females. Fluorescence competitive binding assays revealed that 5 out of 25 candidate volatiles are strong binders (Ki < 10 µM). Behavioral assays revealed that nonadecane and 2-tridecanone, which have high binding affinities in fluorescence competition-binding assays, displayed strong attractiveness to N. lugens. Pursuing this further, molecular docking analysis identified key amino acid residues involved in binding volatile compounds. Overall, our data provide a base for further investigation of the potential physiological functions of CSP3 in Nilaparvata lugens, and extend the function of NlugCSP3 in chemoreception of N. lugens.