Variations in the expression of odorant binding and chemosensory proteins in the developmental stages of whitefly Bemisia tabaci Asia II-1.

The cotton whitefly, Bemisia tabaci, is considered as a species complex with 46 cryptic species, with Asia II-1 being predominant in Asia. This study addresses a significant knowledge gap in the characterization of odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) in Asia II-1. We explored the expression patterns of OBPs and CSPs throughout their developmental stages and compared the motif patterns of these proteins. Significant differences in expression patterns were observed for the 14 OBPs and 14 CSPs of B. tabaci Asia II-1, with OBP8 and CSP4 showing higher expression across the developmental stages. Phylogenetic analysis reveals that OBP8 and CSP4 form distinct clades, with OBP8 appearing to be an ancestral gene, giving rise to the evolution of other odorant-binding proteins in B. tabaci. The genomic distribution of OBPs and CSPs highlights gene clustering on the chromosomes, suggesting functional conservation and evolutionary events following the birth-and-death model. Molecular docking studies indicate strong binding affinities of OBP8 and CSP4 with various odour compounds like ß-caryophyllene, α-pinene, ß-pinene and limonene, reinforcing their roles in host recognition and reproductive functions. This study elaborates on our understanding of the putative roles of different OBPs and CSPs in B. tabaci Asia II-1, hitherto unexplored. The dynamics of the expression of OBPs and CSPs and their interactions with odour compounds offer scope for developing innovative methods for controlling this global invasive pest.

The novel function of an orphan pheromone receptor reveals the sensory specializations of two potential distinct types of sex pheromones in noctuid moth.

Sex pheromones play crucial role in mating behavior of moths, involving intricate recognition mechanisms. While insect chemical biology has extensively studied type I pheromones, type II pheromones remain largely unexplored. This study focused on Helicoverpa armigera, a representative species of noctuid moth, aiming to reassess its sex pheromone composition. Our research unveiled two previously unidentified candidate type II sex pheromones-3Z,6Z,9Z-21:H and 3Z,6Z,9Z-23:H-in H. armigera. Furthermore, we identified HarmOR11 as an orphan pheromone receptor of 3Z,6Z,9Z-21:H. Through AlphaFold2 structural prediction, molecular docking, and molecular dynamics simulations, we elucidated the structural basis and key residues governing the sensory nuances of both type I and type II pheromone receptors, particularly HarmOR11 and HarmOR13. This study not only reveals the presence and recognition of candidate type II pheromones in a noctuid moth, but also establishes a comprehensive structural framework for PRs, contributing to the understanding of connections between evolutionary adaptations and the emergence of new pheromone types.

Structure information analysis and relative content determination of protein and chitin from yellow mealworm larvae using Raman spectroscopy.

Insect protein extract is one of the high-quality protein sources and is frequently viewed as a potential nutrition alternative. However, a more precise method for protein measurement is still needed due to protein overestimation by the Kjeldahl method due to the presence of a large amount of chitin in insects. Therefore, we demonstrated the monitoring of chitin and protein extracted from yellow mealworm larvae through the information on molecular vibration obtained using Raman spectroscopy and infrared (IR) spectroscopy. The NH vibration at 3475 cm-1 is the characteristic peak of chitin in defatted product observed in the Raman spectra. The nitrogen-to-protein conversion factor in protein extracted from larvae by the Raman method was determined based on the NH vibration and found to be 5.66 ± 0.01. We also compared these experimental data to theoretical Raman and IR spectra and determined the possible reasons for why nitrogen elements in chitin affect the determination of protein content. The method of sequentially removing fat and protein could provide more accurate quantification of protein and chitin. Raman spectroscopy is feasible for various types of insects with high chitin content. Compared with the Kjeldahl method, the Raman method is a faster and more accurate measurement method. Moreover, it provides the content of impurities, purity, and structural information.

Structural basis for odorant recognition of the insect odorant receptor OR-Orco heterocomplex.

Insects detect and discriminate a diverse array of chemicals using odorant receptors (ORs), which are ligand-gated ion channels comprising a divergent odorant-sensing OR and a conserved odorant receptor co-receptor (Orco). In this work, we report structures of the ApOR5-Orco heterocomplex from the pea aphid Acyrthosiphon pisum alone and bound to its known activating ligand, geranyl acetate. In these structures, three ApOrco subunits serve as scaffold components that cannot bind the ligand and remain relatively unchanged. Upon ligand binding, the pore-forming helix S7b of ApOR5 shifts outward from the central pore axis, causing an asymmetrical pore opening for ion influx. Our study provides insights into odorant recognition and channel gating of the OR-Orco heterocomplex and offers structural resources to support development of innovative insecticides and repellents for pest control.

Response Surface Methodology (RSM) for Optimizing Protein Extraction from Housefly (Musca domestica) Larvae Fed with Toad and Its Structural Characterization.

With the global population on the rise, an escalating interest exists in environmentally sustainable and friendly protein sources. Insects have emerged as multifaceted resources, viewed not only as potential food items, but also as sources of traditional medicines and proteins. This study utilized response surface methodology (RSM) to ascertain the optimal extraction conditions for proteins from Musca domestica used in toad feeding, denoted as MDPs-T. The yield of MDPs-T was elevated to 18.3% ± 0.2% under these optimized conditions. Subsequently, the particle size, ζ-potentials, and structures of MDPs-T were analyzed and compared with the proteins derived from Musca domestica fed on a normal diet (MDPs-ND). This comparative analysis utilized a range of advanced techniques, involving UV spectroscopy, Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), high-performance gel permeation chromatography (HPGPC), and scanning electron microscopy (SEM). The outcomes have revealed a marginal disparity in the physical and chemical properties between MDPs-T and MDPs-ND. Derosination led to a reduction in the particle size of the MDPs by 10.98% to 62.81%. MDPs-T exhibited a higher proportion of low-molecular-weight components relative to MDPs-ND. Additionally, in a comparative analysis of amino acids, MDPs-T displayed a greater abundance of essential and total amino acids relative to MDPs-ND. Consequently, MDPs-T holds potential as a valuable food supplement for human consumption or as a nutrient-rich feed supplement for animals.

Pierisin, Cytotoxic and Apoptosis-Inducing DNA ADP-Ribosylating Protein in Cabbage Butterfly.

Pierisin-1 was serendipitously discovered as a strong cytotoxic and apoptosis-inducing protein from pupae of the cabbage butterfly Pieris rapae against cancer cell lines. This 98-kDa protein consists of the N-terminal region (27 kDa) and C-terminal region (71 kDa), and analysis of their biological function revealed that pierisin-1 binds to cell surface glycosphingolipids on the C-terminal side, is taken up into the cell, and is cleaved to N- and C-terminal portions, where the N-terminal portion mono-ADP-ribosylates the guanine base of DNA in the presence of NAD to induce cellular genetic mutation and apoptosis. Unlike other ADP-ribosyltransferases, pieisin-1 was first found to exhibit DNA mono-ADP-ribosylating activity and show anti-cancer activity in vitro and in vivo against various cancer cell lines. Pierisin-1 was most abundantly produced during the transition from the final larval stage to the pupal stage of the cabbage butterfly, and this production was regulated by ecdysteroid hormones. This suggests that pierisn-1 might play a pivotal role in the process of metamorphosis. Moreover, pierisin-1 could contribute as a defense factor against parasitization and microbial infections in the cabbage butterfly. Pierisin-like proteins in butterflies were shown to be present not only among the subtribe Pierina but also among the subtribes Aporiina and Appiadina, and pierisin-2, -3, and -4 were identified in these butterflies. Furthermore, DNA ADP-ribosylating activities were found in six different edible clams. Understanding of the biological nature of pierisin-1 with DNA mono-ADP-ribosylating activity could open up exciting avenues for research and potential therapeutic applications, making it a subject of great interest in the field of molecular biology and biotechnology.

UDP-Glycosyltransferases UGT350C3 and UGT344L7 Confer Tolerance to Neonicotinoids in Field Populations of Aphis gossypii.

The cotton aphid, Aphis gossypii, is a polyphagous pest that stunts host plant growth via direct feeding or transmitting plant virus. Due to the long-term application of insecticides, A. gossypii has developed different levels of resistance to numerous insecticides. We found that five field populations had evolved multiple resistances to neonicotinoids. To explore the resistance mechanism mediated by uridine diphosphate glycosyltransferases (UGTs), two upregulated UGT genes in these five strains, UGT350C3 and UGT344L7, were selected for functional analysis of their roles in neonicotinoid detoxification. Transgenic Drosophila bioassay results indicated that compared with the control lines, the UGT350C3 and UGT344L7 overexpression lines were more tolerant to thiamethoxam, imidacloprid, and dinotefuran. Knockdown of UGT350C3 and UGT344L7 significantly increased A. gossypii sensitivity to thiamethoxam, imidacloprid, and dinotefuran. Molecular docking analysis demonstrated that these neonicotinoids could bind to the active pockets of UGT350C3 and UGT344L7. This study provides functional evidence of neonicotinoid detoxification mediated by UGTs and will facilitate further work to identify strategies for preventing the development of neonicotinoid resistance in insects.

A new trypsin inhibitor from Centrosema plumieri effective against digestive proteases from Tribolium castaneum, an eco-friendly alternative.

Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.

In-Depth Structural Simplification of Celangulin V: Design, Synthesis, and Biological Activity.

Celangulin V is a novel botanical insecticide with significant bioactivity and a unique molecular target, but its complex polyol ester structure hinders its broader application in agriculture. To discover new analogues of celangulin V with a simpler structure and enhanced biological activities, we initiated a research project aimed at simplifying its structure and assessing insecticidal efficacy. In this study, a series of novel 1-tetralone derivatives were designed via a structure-based rational design approach and synthesized by a facile method. The biological activities of the target compounds were determined against Mythimna separata (M. separata), Plutella xylostella, and Rhopalosiphum padi. The results revealed that most of the synthesized compounds exhibited superior activities compared to celangulin V. Remarkably, the insecticidal activity of compound 6.16 demonstrated 102-fold greater stomach toxicity than celangulin V against M. separata. In addition, certain compounds showed significant contact toxicity against M. separata, a finding not reported previously in the structural optimization studies of celangulin V. Molecular docking analysis illustrated that the binding pocket of compound 6.16 with the H subunit of V-ATPase was the same as celangulin V. This study presents novel insights into the structural optimization of botanical pesticides.

Structural and functional impacts of neonicotinoids analogues on Apis mellifera's chemosensory protein: Insights from spectroscopic and molecular modeling investigations.

In the intricate web of ecological relationships, pollinators such as the Italian honeybee (Apis mellifera) play a crucial role in maintaining biodiversity and agricultural productivity. This study focuses on the interactions between three neonicotinoid compounds and the honeybee's chemosensory protein 3 (CSP3), a key player in their olfactory system. Employing advanced spectroscopic techniques and molecular modeling, we explore the binding dynamics and conformational changes in CSP3 upon exposure to these pesticides. The research reveals that all three neonicotinoids considerably quench CSP3's fluorescence through a dynamic and static mixing mechanism, indicating a strong binding affinity, predominantly driven by hydrophobic interactions. UV-visible absorption, synchronous fluorescence, and 3D fluorescence spectra support slight changes in the microenvironment around the aromatic amino acids of CSP3. Circular dichroism spectra indicate a reduction in CSP3's α-helix content, suggesting structural alterations. Molecular docking and dynamics simulations further elucidate the binding modes and stability of these interactions, highlighting the role of specific amino acids in CSP3's binding cavity. Findings provide critical insights into molecular mechanisms by which neonicotinoids may impair honeybee chemosensory function, offering implications for designing safer pesticides and understanding the broader ecological impact of these chemicals on pollinator health.

Trial and error: New insights into recombinant expression of membrane-bound insect cytochromes P450 in Escherichia coli systems.

Insect cytochromes P450 (CYP450s) are key enzymes responsible for a wide array of oxidative transformations of both endogenous and exogenous substrates. However, there is currently no a universal guideline established for heterologous expression of membrane-bound CYP450s, which hampers their downstream biochemical and structural studies. In this study, we conducted large-scale screening of protein overexpression in Escherichia coli using 71 insect CYP450 sequences and optimized the expression of a difficult-to-express CYP450 (CYP6HX3) using eight different optimizations, including selection of host strains and expression vectors, alternative of leader signal peptides, and N-terminal modifications. We confirmed that 1) Only insect CYP450s belonging to the CYP347 family could be expressed with N-terminal fusion of ompA2+ signal peptide in E. coli expression system. 2) E. coli Lemo 21 (DE3) effectively improved the expression of CYP6HX3 in the plasma membrane. 3) A brick-red appearance occurred frequently in the expressed thallus or membrane proteins, but this phenomenon could not necessarily indicate successful overexpression of target CYP450s. These findings provide new insights into the recombinant expression of insect CYP450s in E. coli systems and will facilitate the theoretical approaches for functional expression and production of eukaryotic CYP450s.

Structural basis of odor sensing by insect heteromeric odorant receptors.

Most insects, including human-targeting mosquitoes, detect odors through odorant-activated ion channel complexes consisting of a divergent odorant-binding subunit (OR) and a conserved co-receptor subunit (Orco). As a basis for understanding how odorants activate these heteromeric receptors, we report here cryo-electron microscopy structures of two different heteromeric odorant receptor complexes containing ORs from disease-vector mosquitos Aedes aegypti or Anopheles gambiae. These structures reveal an unexpected stoichiometry of one OR to three Orco subunits. Comparison of structures in odorant-bound and unbound states indicates that odorant binding to the sole OR subunit is sufficient to open the channel pore, suggesting a mechanism of OR activation and a conceptual framework for understanding evolution of insect odorant receptor sensitivity.

Molecular Characterization of Chemosensory Protein (CSP) Genes and the Involvement of AgifCSP5 in the Perception of Host Location in the Aphid Parasitoid Aphidius gifuensis.

Aphidius gifuensis is the dominant parasitic natural enemy of aphids. Elucidating the molecular mechanism of host recognition of A. gifuensis would improve its biological control effect. Chemosensory proteins (CSPs) play a crucial role in insect olfactory systems and are mainly involved in host localization. In this study, a total of nine CSPs of A. gifuensis with complete open reading frames were identified based on antennal transcriptome data. Phylogenetic analysis revealed that AgifCSPs were mainly clustered into three subgroups (AgifCSP1/2/7/8, AgifCSP3/9, and AgifCSP4/5/6). AgifCSP2/5 showed high expression in the antennae of both sexes. Moreover, AgifCSP5 was found to be specifically expressed in the antennae. In addition, fluorescent binding assays revealed that AifCSP5 had greater affinities for 7 of 32 volatile odor molecules from various sources. Molecular docking and site-directed mutagenesis results revealed that the residue at which AgifCSP5 binds to these seven plant volatiles is Tyr75. Behavior tests further confirmed that trans-2-nonenal, one of the seven active volatiles in the ligand binding test, significantly attracted female adults at a relatively low concentration of 10 mg/mL. In conclusion, AgifCSP5 may be involved in locating aphid-infested crops from long distances by detecting and binding trans-2-nonenal. These findings provide a theoretical foundation for further understanding the olfactory recognition mechanisms and indirect aphid localization behavior of A. gifuensis from long distances by first identifying the host plant of aphids.

Changes in physicochemical, structural and functional properties, and lysinoalanine formation during the unfolding and refolding of pH-shifted black soldier fly larvae albumin.

The changes of physicochemical, structural and functional properties and the lysinoalanine (LAL) formation during the unfolding and refolding of black soldier fly larvae albumin (BSFLA) induced by acid/alkaline pH shift were explored. The results showed that acid/alkaline conditions induced unfolding of BSFLA structure, but also accompanied by the formation of some large aggregates due to the hydrophobic interactions, hydrogen bonds, and disulfide bonds. Compared with control or pH1.5 shift, pH12 shift treatment significantly increased the electrostatic repulsion, surface hydrophobicity, free sulfhydryl group, and deamidation reactions, but reduced the fluorescence intensity of BSFLA, and these change in protein conformation contributed to increase in solubility, emulsion activity, and emulsion stability. But the content of LAL in BSFLA was increased by 93.39 % by pH 12 shift treatment. In addition, pH1.5 shift modified BSFLA tended to form ß-sheet structure through unfolding and refolding, resulting in the formation of aggregates with larger particle sizes, and reducing the solubility and the LAL content by 7.93 % and 65.53 %, respectively. SDS-PAGE profile showed that pH12/1.5 shifting did not cause irreversible denaturation of protein molecules. Therefore, pH12-shift is good way to improve the functional properties of BSFLA, but the content of LAL should be reduced to make it better used in food.

Production and Release of Proinflammatory Mediators by the Cockroach Allergen Bla g 1 via a Shared Membrane-Destabilization Mechanism.

The cockroach allergen Bla g 1 encloses an exceptionally large hydrophobic cavity, which allows it to bind and deliver unsaturated fatty acid ligands. Bla g 1-mediated delivery of naturally occurring (nMix) ligands has been shown to destabilize lipid membranes, contributing to its digestive/antiviral functions within the source organism. However, the consequences of this activity on Bla g 1 allergenicity following human exposure remain unknown. In this work, we show that Bla g 1-mediated membrane disruption can induce a proinflammatory immune response in mammalian cells via two complementary pathways. At high concentrations, the cytotoxic activity of Bla g 1 induces the release of proinflammatory cytosolic contents including damage-associated molecular patterns (DAMPs) such as heat-shock Protein-70 (HSP70) and the cytokine interleukin-1 (IL-1ß). Sublytic concentrations of Bla g 1 enhanced the ability of phospholipase A2 (PLA2) to extract and hydrolyze phospholipid substrates from cellular membranes, stimulating the production of free polyunsaturated fatty acids (PUFAs) and various downstream inflammatory lipid mediators. Both of these effects are dependent on the presence of Bla g 1's natural fatty-acid (nMix) ligands with CC50 values corresponding to the concentrations required for membrane destabilization reported in previous studies. Taken together, these results suggest that mechanisms through which Bla g 1-mediated lipid delivery and membrane destabilization could directly contribute to cockroach allergic sensitization.

Enhanced sensitivity of chimeric insect olfactory co-receptors for detecting odorant molecules.

Insect olfactory receptors (ORs) are seven-transmembrane domain ion channels that function by forming heteromeric complexes with olfactory receptor co-receptors (Orcos). In this study, we investigated the potential for enhancing sensitivity of odor detection and responsivity through genetic modification of Orcos, considering its wider application in odor sensing. First, we measured the intensity of response to 1-octen-3-ol for the mosquito Aedes aegypti OR (AaOR8) when complexed individually with an Orco from the same mosquito (AaOrco), the honeybee Apis mellifera (AmOrco), the silkworm Bombyx mori (BmOrco), or the fruit fly Drosophila melanogaster (DmOrco). Relative to the other Orcos, AmOrco demonstrated higher sensitivity and responsivity, with a 1.8 to 21-fold decrease in the half-maximal effective concentration (EC50) and a 1.6-8.8-fold increase in the maximal effect (Emax), respectively. Furthermore, AmOrco co-expressed with AaOR10, BmOR56, or DmOR47a showed higher sensitivity and responsivity than AaOrco, BmOrco, or DmOrco co-expressed with their respective ORs. To further increase sensitivity and responsivity, we engineered chimeric Orcos by fusing AmOrco with DmOrco, considering the domain characteristics of Orcos. The response to 1-octen-3-ol was evaluated for AaOR8 when complexed individually with AmOrco, as well as for a mutant that combines DmOrco from the N-terminal (NT) to the C-terminal region of the fourth transmembrane domain (TM4) with the region of AmOrco following TM4 (Dm[NT-TM4]AmOrco). When compared to AmOrco, Dm(NT-TM4)AmOrco showed higher sensitivity and responsivity, with a 1.4-fold decrease in the EC50 and a 1.4-fold increase in the Emax, respectively. In addition, Dm(NT-TM4)AmOrco co-expressed with either DmOR47a or BmOR56 demonstrated higher sensitivity and responsivity than AmOrco co-expressed with their respective ORs. These results suggest that AmOrco could be a relatively more sensitive Orco, and further enhancement of sensitivity and responsivity could be achieved through recombination with heterologous Orcos near the TM4 of AmOrco.

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.

Increasing protein identifications in bottom-up proteomics of T. castaneum - Exploiting synergies of protein biochemistry and bioinformatics.

Depending on the respective research question, LC-MS/MS based bottom-up proteomics poses challenges from the initial biological sample all the way to data evaluation. The focus of this study was to investigate the influence of sample preparation techniques and data analysis parameters on protein identification in Tribolium castaneum by applying free software proteomics platform Max Quant. Multidimensional protein extraction strategies in combination with electrophoretic or chromatographic off-line protein pre-fractionation were applied to enhance the spectrum of isolated proteins from T. castaneum and reduce the effect of co-elution and ion suppression effects during nano-LC-MS/MS measurements of peptides. For comprehensive data analysis, MaxQuant was used for protein identification and R for data evaluation. A wide range of parameters were evaluated to gain reproducible, reliable, and significant protein identifications. A simple phosphate buffer, pH 8, containing protease and phosphatase inhibitor cocktail and application of gentle extraction conditions were used as a first extraction step for T.castaneum proteins. Furthermore, a two-dimensional extraction procedure in combination with electrophoretic pre-fractionation of extracted proteins and subsequent in-gel digest resulted in almost 100% increase of identified proteins when compared to chromatographic fractionation as well as one-pot-analysis. The additionally identified proteins could be assigned to new molecular functions or cell compartments, emphasizing the positive effect of extended sample preparation in bottom-up proteomics. Besides the number of peptides during post-processing, MaxQuant's Match between Runs exhibited a crucial effect on the number of identified proteins. A maximum relative standard deviation of 2% must be considered for the data analysis. Our work with Tribolium castaneum larvae demonstrates that sometimes - depending on matrix and research question - more complex and time-consuming sample preparation can be advantageous for isolation and identification of additional proteins in bottom-up proteomics.

Exploring the binding affinity and characteristics of DcitOBP9 in citrus psyllids.

Odorant-binding proteins (OBPs) are crucial in insect olfaction. The most abundant expressed OBP of citrus psyllids, DcitOBP9 encodes 148 amino acids. DcitOBP9 lacks a transmembrane structure and possesses a 17-amino acid signal peptide at the N-terminus. Characterized by the six conserved cysteine sites, DcitOBP9 is classified as the Classical-OBP family. RT-qPCR experiments revealed ubiquitous expression of DcitOBP9 across all developmental stages of the citrus psyllid, with predominant expression in adults antennae. Fluorescence competitive binding assays demonstrated DcitOBP9's strong affinity for ocimene, linalool, dodecanoic acid, and citral, and moderate affinity for dimethyl trisulfide. Additionally, it binds to myrcia, (-)-trans-caryophyllene, (±)-Citronellal, nonanal, and (+)-α-pinene. Among them, ocimene, linalool, and dodecanoic acid were dynamically bound to DcitOBP9, while citral was statically bound to DcitOBP9. Molecular docking simulations with the top five ligands indicated that amino acid residues V92, S72, P128, L91, L75, and A76 are pivotal in the interaction between DcitOBP9 and these odorants. These findings suggest DcitOBP9's involvement in the citrus psyllid's host plant recognition and selection behaviors, thereby laying a foundation for elucidating the potential physiological and biological functions of DcitOBP9 and developing attractants.

In silico identification and expression analysis of superoxide dismutases in Tenebrio molitor.

BACKGROUND: Insects encounter various environmental stresses, in response to which they generate reactive oxygen species (ROS). Superoxide dismutase (SOD) is an antioxidant metalloenzyme that scavenges superoxide radicals to prevent oxidative damage. OBJECTIVE: To investigate expressions of SODs under oxidative stress in Tenebrio molitor. METHODS: Here, we investigated the transcriptional expression of SODs by pesticide and heavy metals in Tenebrio moltior. First, we searched an RNA-Seq database for T. molitor SOD (TmSOD) genes and identified two SOD isoforms (TmSOD1-iso1 and iso2). We examined their activities under developmental stage, tissue-specific, and various types (pesticide and heavy metal) of oxidative stress by using qPCR. RESULTS: Our results revealed two novel forms of TmSODs. These TmSODs had a copper/zinc superoxide dismutase domain, active site, Cu2+ binding site, Zn2+ binding site, E-class dimer interface, and P-class dimer interface. TmSODs (TmSOD1-iso1 and iso2) were expressed in diverse developmental phases and tissues. Pesticides and heavy metals caused an upregulation of these TmSODs. CONCLUSION: Our findings suggest that the two TmSODs have different functions in T. molitor, providing insights into the detoxification ability of T. molitor.