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Simple Summary: The swallowtail butterfly Papilio xuthus is a specialized herbivorous insect, with its larvae solely utilizing the Rutaceae plants as hosts. The host seeking and orientation of this butterfly mainly rely on chemosensory-related genes expressed in the antennae, including chemosensory proteins (CSPs). Here, we characterized the putative roles of a female-antenna-biased PxutCSP19 in P. xuthus associated with the perception of host volatiles and the sequestering of insecticides. This study examined the key functions of PxutCSP19 in the interactions of six host-derived odorants and eight insecticides. Notably, an extended N-terminus of PxutCSP19 did not significantly affect the binding specificities of this protein to ligands with high affinities. Our findings provide insight into the binding mechanisms of PxutCSP19 in P. xuthus to host volatiles and insecticides. Chemosensory protein (CSP) genes significantly enriched in the female antennae are potential molecular candidates for mediating female oviposition behaviors. In this study, we presented the interaction mechanisms of a female-antenna-biased PxutCSP19 in Papilio xuthus to 47 host volatiles, four biopesticides and 24 synthetic insecticides. Using a bioinformatics-based homology search, 22 genes orthologous to PxutCSP19 were identified from 22 other Papilio butterflies with high sequence identities to each other (73.20~98.72%). Multiple alignment analyses revealed a particularly extended N-terminus of Papilio CSP19s (an average of 154 residues) compared to insects' typical CSPs (approximately 120 residues). The expression profiles indicated that PxutCSP19 was significantly enriched in the female antennae, with a 31.81-fold difference relative to the male antennae. In ligand-binding assays, PxutCSP19 could strongly bind six host odorants with high affinities, ranging from dissociation constant (Ki) values of 20.44 ± 0.64 μM to 22.71 ± 0.73 μM. Notably, this protein was tuned to a monoterpenoid alcohol, linalool, which generally existed in the Rutaceae plants and elicited electrophysiological and behavioral activities of the swallowtail butterfly. On the other hand, PxutCSP19 was also capable of binding eight insecticides with stronger binding abilities (Ki < 12 μM) compared to host odorants. When an extended N-terminal region of PxutCSP19 was truncated into two different proteins, they did not significantly affect the binding of PxutCSP19 to ligands with high affinities, suggesting that this extended N-terminal sequences were not involved in the specificity of ligand recognition. Altogether, our study sheds light on the putative roles of PxutCSP19 enriched in the female antennae of P. xuthus in the perception of host volatiles and the sequestering of insecticides, and it complements the knowledge of butterfly CSPs in olfaction and insecticide resistance. [ABSTRACT FROM AUTHOR]

Abstract 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 β-caryophyllene, α-pinene, β-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.

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. [ABSTRACT FROM AUTHOR]

Chemosensory proteins (CSPs) are highly efficient carry tools to bind and deliver hydrophobic compounds, which play an important role in the chemosensory process in insects. The diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), is a cosmopolitan pest that attacks cruciferous crops. However, the detailed physiological functions of CSPs in P. xylostella remain limited to date. Here, we identified a typical CSP, named Pxyl CSP18, in P. xylostella and investigated its expression patterns and binding properties of volatiles. PxylCSP18 was highly expressed in antennae and head (without antennae), and the expression level in the male antennae of P. xylostella was obviously higher than that in the female antennae. Moreover, Pxyl CSP18 has a relatively broad binding spectrum. Fluorescence competitive binding assays showed that Pxyl CSP18 had strong binding abilities with 14 plant volatiles (Ki < 10 μM) that were repellent or attractive to P. xylostella. Notably, Pxyl CSP18 had no significant binding affinity to (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, and (Z)-11-hexadecenyl alcolol, which are the pheromone components of P. xylostella. The attractive effects of trans-2-hexen-1-ol and isopropyl isothiocyanate to male adults and the attractive effects of isopropyl isothiocyanate and the repellent effects of linalool to female adults were significantly decreased after knocked down the expression of PxylCSP18. Our results revealed that Pxyl CSP18 might play an important role in host plant detection, avoidance of unsuitable hosts, and selection of oviposition sites; however, it does not participate in mating behavior. Overall, these results extended our knowledge on the CSP-related functions, which provided insightful information about CSP-targeted insecticides. [ABSTRACT FROM AUTHOR]

Chemosensory protein (CSP) genes significantly enriched in the female antennae are potential molecular candidates for mediating female oviposition behaviors. In this study, we presented the interaction mechanisms of a female-antenna-biased PxutCSP19 in Papilio xuthus to 47 host volatiles, four biopesticides and 24 synthetic insecticides. Using a bioinformatics-based homology search, 22 genes orthologous to PxutCSP19 were identified from 22 other Papilio butterflies with high sequence identities to each other (73.20~98.72%). Multiple alignment analyses revealed a particularly extended N-terminus of Papilio CSP19s (an average of 154 residues) compared to insects’ typical CSPs (approximately 120 residues). The expression profiles indicated that PxutCSP19 was significantly enriched in the female antennae, with a 31.81-fold difference relative to the male antennae. In ligand-binding assays, PxutCSP19 could strongly bind six host odorants with high affinities, ranging from dissociation constant (Ki) values of 20.44 ± 0.64 μM to 22.71 ± 0.73 μM. Notably, this protein was tuned to a monoterpenoid alcohol, linalool, which generally existed in the Rutaceae plants and elicited electrophysiological and behavioral activities of the swallowtail butterfly. On the other hand, PxutCSP19 was also capable of binding eight insecticides with stronger binding abilities (Ki < 12 μM) compared to host odorants. When an extended N-terminal region of PxutCSP19 was truncated into two different proteins, they did not significantly affect the binding of PxutCSP19 to ligands with high affinities, suggesting that this extended N-terminal sequences were not involved in the specificity of ligand recognition. Altogether, our study sheds light on the putative roles of PxutCSP19 enriched in the female antennae of P. xuthus in the perception of host volatiles and the sequestering of insecticides, and it complements the knowledge of butterfly CSPs in olfaction and insecticide resistance.

Simple Summary: A previous study found that an indigenous endoparasitic wasp Cotesia ruficrus, has a good control effect on the invasive pest Spodoptera frugiperda. Understanding the molecular mechanism of host recognition of C. ruficrus would help improve its biological control effect. Chemoreceptor proteins (CSPs) play a crucial role in insect olfactory perception. The CrufCSP1 protein was obtained via prokaryotic expression, and its binding properties were evaluated using fluorescence binding assays. Molecular docking was used to verify the function of CrufCSP1. The findings revealed that CrufCSP1 has the ability to bind with seven host-related compounds. This information serves as a crucial reference for developing natural enemy attractants for pest control. Chemosensory proteins (CSPs) are a class of soluble proteins that facilitate the recognition of chemical signals in insects. While CSP genes have been identified in many insect species, studies investigating their function remain limited. Cotesia ruficrus (Hymenoptera: Braconidae) holds promise as an indigenous biological control agent for managing the invasive pest Spodoptera frugiperda (Lepidoptera: Noctuidae) in China. This study aimed to shed light on the gene expression, ligand binding, and molecular docking of CrufCSP1 in C. ruficrus. A RT-qPCR analysis revealed that the expression of CrufCSP1 was higher in the wings, with male adults exhibiting significantly higher relative expression levels than other developmental stages. A fluorescence competitive binding analysis further demonstrated that CrufCSP1 has a high binding ability with several host-related volatiles, with trans-2-hexenal, octanal, and benzaldehyde showing the strongest affinity to CrufCSP1. A molecular docking analysis indicated that specific amino acid residues (Phe24, Asp25, Thr53, and Lys81) of CrufCSP1 can bind to these specific ligands. Together, these findings suggest that CrufCSP1 may play a crucial role in the process of C. ruficrus locating hosts. This knowledge can contribute to the development of more efficient and eco-friendly strategies for protecting crops and managing pests. [ABSTRACT FROM AUTHOR]

Aphidoletes aphidimyza is a predator that is an important biological agent used to control agricultural and forestry aphids. Although many studies have investigated its biological and ecological characteristics, few molecular studies have been reported. The current study was performed to identify suitable reference genes to facilitate future gene expression and function analyses via quantitative reverse transcription PCR. Eight reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RPS13, RPL8, RPS3, α-Tub, β-actin, RPL32, and elongation factor 1 alpha (EF1-α) were selected. Their expression levels were determined under four different experimental conditions (developmental stages, adult tissues, sugar treatment, and starvation treatment) using qRT-PCR technology. The stability was evaluated with five methods (Ct value, geNorm, NormFinder, BestKeeper, and RefFinder). The results showed that GAPDH, RPL32, and EF1-α were ranked as the best reference gene combinations for measuring gene expression levels among different developing stages and in various starvation treatments. RPL8 and RPS3 were recommended to normalize the gene expression levels among different adult tissues. RPL32, β-actin, and EF1-α were recommended sugar-feeding conditions. To validate the utility of the selected reference pair, RPL8, and RPS3, we estimated the tissue-biased expression level of a chemosensory protein gene (AaphCSP1). As expected, AaphCSP1 is highly expressed in the antennae and lowly expressed in the abdomen. These findings will lay the foundation for future research on the molecular physiology and biochemistry of A. aphidimyza. [ABSTRACT FROM AUTHOR]

Summary: Aphid salivary proteins are critical in modulating plant defence responses. Grain aphid Sitobion miscanthi is an important wheat pest worldwide. However, the molecular basis for the regulation of the plant resistance to cereal aphids remains largely unknown. Here, we show that SmCSP4, a chemosensory protein from S. miscanthi saliva, is secreted into wheat plants during aphid feeding. Delivery of SmCSP4 into wheat leaves activates salicylic acid (SA)‐mediated plant defence responses and subsequently reduces aphid performance by deterring aphid feeding behaviour. In contrast, silencing SmCSP4 gene via nanocarrier‐mediated RNAi significantly decreases the ability of aphids to activate SA defence pathway. Protein–protein interaction assays showed that SmCSP4 directly interacts with wheat transcriptional factor TaWRKY76 in plant nucleus. Furthermore, TaWRKY76 directly binds to the promoter of SA degradation gene Downy Mildew Resistant 6 (DMR6) and regulates its gene expression as transcriptional activator. SmCSP4 secreted by aphids reduces the transcriptional activation activity of TaWRKY76 on DMR6 gene expression, which is proposed to result in increases of SA accumulation and enhanced plant immunity. This study demonstrated that SmCSP4 acts as salivary elicitor that is involved in activating SA signalling defence pathway of wheat by interacting with TaWRKY76, which provide novel insights into aphid‐cereal crops interactions and the molecular mechanism on induced plant immunity. [ABSTRACT FROM AUTHOR]

Chemosensory proteins (CSPs) were necessary for insect sensory system to perform important processes such as feeding, mating, spawning, and avoiding natural enemies. However, their functions in non-olfactory organs have been poorly studied. To clarify the function of CSPs in the development of Mythimna separata (Walker) larvae, two CSP genes, MsCSP17 and MsCSP18 , were identified from larval integument transcriptome dataset. Both of MsCSP17 and MsCSP18 contained four conserved cysteine sites (C × (6)-C × (18)-C × (2)-C), with a signal peptide at the N-terminal. RT-qPCR analysis showed that MsCSP17 and MsCSP18 have different expression patterns among different developmental stages and tissues. MsCSP17 was highly expressed in 1st–4th instar larvae, and MsCSP18 had high expression in adults. Both genes were expressed highly in larval head, thorax, integument and mandible. Moreover, both of MsCSP17 and MsCSP18 were lowly expressed in larval integuments when larvae molted for 6 h and 9 h from 3rd to 4th instar, but highly at the beginning and end phase during molting. After injection of dsMsCSP17 and dsMsCSP18, the expression levels of two genes decreased significantly, with the body weight of larvae decreased, the mortality increased, and the eclosion rate decreased. It was suggested that MsCSP17 and MsCSP18 contributed to the development of M. separata larvae. [ABSTRACT FROM AUTHOR]

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.

Simple Summary: Since existing control strategies are not yet effective, in order to elucidate the molecular mechanisms of protein–ligand binding for effective pest control, three ligands that bind best to chemosensory protein 2 of Sirex nitobei were screened in this study. The host plant volatile (+)-α-pinene, symbiotic fungal volatiles terpene and (−)-globulol were found to bind most stably by molecular docking and dynamic simulations, and their free binding energies were calculated by the molecular mechanics Poisson–Boltzmann surface area method. Furthermore, some key amino acid residues were deeply explored, providing a favorable molecular basis for regulating the behavioral interactions of insects and developing new pest control strategies. Sirex nitobei is an important wood-boring wasp to conifers native to Asia, causing considerable economic and ecological damage. However, the current control means cannot achieve better efficiency, and it is expected to clarify the molecular mechanism of protein–ligand binding for effective pest control. This study analyzed the expression pattern of CSP2 in S. nitobei (SnitCSP2) and its features of binding to the screened ligands using molecular docking and dynamic simulations. The results showed that SnitCSP2 was significantly expressed in female antennae. Molecular docking and dynamic simulations revealed that SnitCSP2 bound better to the host plant volatile (+)-α-pinene and symbiotic fungal volatiles terpene and (−)-globulol than other target ligands. By the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method, the free binding energies of the three complexes were calculated as −44.813 ± 0.189 kJ/mol, −50.446 ± 0.396 kJ/mol, and −56.418 ± 0.368 kJ/mol, and the van der Waals energy was found to contribute significantly to the stability of the complexes. Some key amino acid residues were also identified: VAL13, GLY14, LYS61, MET65, and LYS68 were important for the stable binding of (+)-α-pinene by SnitCSP2, while for terpenes, ILE16, ALA25, TYR26, CYS29, GLU39, THR37, and GLY40 were vital for a stable binding system. We identified three potential ligands and analyzed the interaction patterns of the proteins with them to provide a favorable molecular basis for regulating insect behavioral interactions and developing new pest control strategies. [ABSTRACT FROM AUTHOR]

Chemosensory proteins (CSPs) perform several functions in insects. This study performed the gene expression, ligand-binding, and molecular docking assays on the EforCSP3 identified in the parasitoid wasp Encarsia formosa, to determine whether EforCSP3 functions in olfaction, especially in host location and host preference. The results showed that EforCSP3 was highly expressed in the female head, and its relative expression was much higher in adults than in other developmental stages. The fluorescence binding assays suggested that the EforCSP3 exhibited high binding affinities to a wide range of host-related volatiles, among which dibutyl phthalate, 1-octene, β-elemene, and tridecane had the strongest binding affinity with EforCSP3, besides α-humulene and β-myrcene, and should be assessed as potential attractants. Protein structure modeling and molecular docking predicted the amino acid residues of EforCSP3 possibly involved in volatile binding. α-Humulene and β-myrcene attracted E. formosa in a previous study and exhibited strong binding affinities with EforCSP3 in the current study. In conclusion, EforCSP3 may be involved in semiochemical reception by E. formosa.

BACKGROUND: Chemosensory proteins (CSPs) play a vital role in the response to environmental stimuli in insects. However, the involvement of insect CSPs in the stress response to night‐time environmental changes has not been examined. RESULTS: In the current study, four TiCSP genes were first cloned from Thalassodes immissaria by transcriptome and RACE‐PCR techniques. TiCSPs had typical characteristics of insect CSPs, including a highly conserved four‐cysteine motif and olfactory‐specific protein D (OS‐D) or OS‐D superfamily domains. TiCSP1–4 were clustered classified within different clades in a phylogenetic analysis and were differentially expressed at all developmental stages. Under night‐time artificial light stress, the expression levels of TiCSP1 in males were significantly decreased at 24 h, and those of TiCSP2 were decreased in both adult sexes at 48 h. In a molecular docking analysis, TiCSPs showed relatively higher binding affinities with sex pheromone components than with host plant volatile molecules. CONCLUSION: Taking the reduced expression levels of TiCSPs and binding affinities into account, TiCSP1 and TiCSP2 are involved in the stress response processes of T. immissaria under light treatment. Our study supplies basic data for the evaluation of the effects of light interference control technology – an emerging physical control measure on nontarget pests of lychee orchards. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Chemosensory proteins (CSPs) are a class of soluble proteins that facilitate the recognition of chemical signals in insects. While CSP genes have been identified in many insect species, studies investigating their function remain limited. Cotesia ruficrus (Hymenoptera: Braconidae) holds promise as an indigenous biological control agent for managing the invasive pest Spodoptera frugiperda (Lepidoptera: Noctuidae) in China. This study aimed to shed light on the gene expression, ligand binding, and molecular docking of CrufCSP1 in C. ruficrus. A RT-qPCR analysis revealed that the expression of CrufCSP1 was higher in the wings, with male adults exhibiting significantly higher relative expression levels than other developmental stages. A fluorescence competitive binding analysis further demonstrated that CrufCSP1 has a high binding ability with several host-related volatiles, with trans-2-hexenal, octanal, and benzaldehyde showing the strongest affinity to CrufCSP1. A molecular docking analysis indicated that specific amino acid residues (Phe24, Asp25, Thr53, and Lys81) of CrufCSP1 can bind to these specific ligands. Together, these findings suggest that CrufCSP1 may play a crucial role in the process of C. ruficrus locating hosts. This knowledge can contribute to the development of more efficient and eco-friendly strategies for protecting crops and managing pests.

Chemosensory proteins (CSPs) are important molecular components of the insect olfactory system, which are involved in capturing, binding, and transporting hydrophobic odour molecules across the sensillum in sensillar lymph in regulating insect behavior. This protein family (CSPs) is also involved in many other systems that are not linked to olfactory receptors in olfactory sensilla. The brown planthopper (BPH) is a monophagous pest of rice that causes damage by sucking phloem sap and transmitting a number of diseases caused by viruses. In this study, fluorescence competitive binding assay and fluorescence quenching assay at acidic pH were performed as well as homology modelling to describe the binding affinity of NlugCSP10. Fluorescence competitive binding assay (FCBA) demonstrated that NlugCSP10 bound strongly to nonadecane, farnesene, and 2-tridecanone at acidic pH. The results of FCBA indicated that NlugCSP10 bound different ligands at the physiological pH (5.0) of the bulk sensillum lymph. Fluorescence quenching assay demonstrated that NlugCSP10 generated a stable complex with 2-tridecanone, while two ligands nonadecane and farnesene collided due to molecular collisions. The interaction of selected ligands with the modelled structure of NlugCSP10 was also analyzed, which found the key amino acids (Gln23, Gln24, Gln25, Asn27, Met33, Ser34, Ile35, Tyr36, Asn42, Met43, Val45, Asn46, Asn93, Arg96, Ala97, Lys99, and Ala100) in NlugCSP10 that were involved in binding of volatile compounds. The present study contributes to the binding profile of NlugCSP10 that promotes the development of behaviorally active ligands based on BPH olfactory system.

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. [Display omitted] • The expression level of RpedCSP22 in both male and female R. pedestris reached its peak on day 7. • RpedCSP22 had optimal binding affinity with three soybean volatiles under neutral conditions. • RpedCSP22 silencing rate could reach more than 90 % by RNAi. • EAG responses of dsCSP22 R. pedestris was significantly lower than that of control group. • The attraction of dsCSP22 R. pedestris toward the three soybean volatiles was significantly attenuated. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Three neonicotinoids bind to bee CSP3 and cause olfactory dysfunction in bees • Hydrophobic interactions drive neonicotinoid-CSP3 binding • Temperature influences the binding dynamics between CSP3 and neonicotinoids • CSP3's α-helix content decreases upon neonicotinoid binding [ABSTRACT FROM AUTHOR]

Insects stimulate specific behaviors by correctly recognizing scent molecules in the external environment. Sirex nitobei, a wood-boring wasp species native to Asia with a distribution area that includes the Palaearctic and Oriental regions, is a significant pest of conifers. Focusing on the molecular mechanism of protein-ligand binding, this study resolved the tissue expression profile of CSP4 from S. nitobei (SnitCSP4) and probed its binding properties with target ligands using molecular docking and dynamics simulations to verify the odor recognition function of this protein. The open reading frame (ORF) of SnitCSP4 was 396 bp, encoding 131 amino acids. Tissue expression analysis revealed that SnitCSP4 was significantly expressed in female antennae and docking showed that all ligands were bound in hydrophobic cavities and close to many hydrophobic amino acid residues. GLN68 and LEU49 were important amino acid residues for SnitCSP4 to bind various odors, and THR9 was the key ligand-binding site in identifying (-)-globulol in the SnitCSP4. Molecular dynamics verified the docking results, confirming that SnitCSP4 bound well to two sex pheromone molecules, three host plant volatiles, and three symbiotic fungal volatiles, with (Z)-7-heptacosene, (Z)-9-nonacosene, and (-)-globulol binding being the most highly stable. These results mean that SnitCSP4 is critical for insects recognizing scent molecules, providing a favorable molecular basis for regulating the behavioral interactions between S. nitobei and the environment, and offering the possibility of developing new strategies for more environmentally friendly and effective control. [ABSTRACT FROM AUTHOR]

Unraveling the origin of molecular pathways underlying the evolution of adaptive traits is essential for understanding how new lineages emerge, including the relative contribution of conserved ancestral traits and newly evolved derived traits. Here, we investigated the evolutionary divergence of sex pheromone communication from moths (mostly nocturnal) to butterflies (mostly diurnal) that occurred ~119 million years ago. In moths, it is the females that typically emit pheromones to attract male mates, but in butterflies males emit pheromones that are used by females for mate choice. The molecular bases of sex pheromone communication are well understood in moths, but they have remained relatively unexplored in butterflies. We used a combination of transcriptomics, real time qPCR, and phylogenetics to identify genes involved in the different steps (i.e., production, regulation, and reception) of sex pheromone communication of the butterfly Bicyclus anynana. Our results show that the biosynthesis and reception of sex pheromones relies both on moth-specific gene families (reductases) and on more ancestral insect gene families (desaturases, olfactory receptors, odorant binding proteins). Interestingly, B. anynana appears to use what was believed to be the moth-specific neuropeptide Pheromone Biosynthesis Activating Neuropeptide (PBAN) for regulating sex pheromone production. Altogether, our results suggest that a mosaic pattern best explains how sex pheromone communication evolved in butterflies, with some molecular components derived from moths, and others conserved from more ancient insect ancestors. This is the first large-scale investigation of the genetic pathways underlying sex pheromone communication in a butterfly. [ABSTRACT FROM AUTHOR]

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The mechanisms of insecticide resistance are complex. Recent studies have revealed a novel mechanism involving the chemosensory system in insecticide resistance. However, the specific binding mechanism between olfactory-related genes and insecticides needs to be clarified. In this study, the binding mechanism between pyrethroid insecticide deltamethrin and RpCSP6 from Rhopalosiphum padi was investigated by using computational and multiple experimental methods. RpCSP6 was expressed in different tissues and developmental stages of R. padi and can be induced by deltamethrin. Knockdown of RpCSP6 significantly increased the susceptibility of R. padi to deltamethrin. The binding affinity of RpCSP6 to 24 commonly used insecticides was measured. Seven key residues were found to steadily interact with deltamethrin, indicating their significance in the binding affinity to the insecticide. Our research provided insights for effectively analyzing the binding mechanism of insect CSPs with insecticides, facilitating the development of new and effective insecticides that target insect CSPs.

Sirex nitobei is an important wood-boring wasp to conifers native to Asia, causing considerable economic and ecological damage. However, the current control means cannot achieve better efficiency, and it is expected to clarify the molecular mechanism of protein–ligand binding for effective pest control. This study analyzed the expression pattern of CSP2 in S. nitobei (SnitCSP2) and its features of binding to the screened ligands using molecular docking and dynamic simulations. The results showed that SnitCSP2 was significantly expressed in female antennae. Molecular docking and dynamic simulations revealed that SnitCSP2 bound better to the host plant volatile (+)-α-pinene and symbiotic fungal volatiles terpene and (−)-globulol than other target ligands. By the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method, the free binding energies of the three complexes were calculated as −44.813 ± 0.189 kJ/mol, −50.446 ± 0.396 kJ/mol, and −56.418 ± 0.368 kJ/mol, and the van der Waals energy was found to contribute significantly to the stability of the complexes. Some key amino acid residues were also identified: VAL13, GLY14, LYS61, MET65, and LYS68 were important for the stable binding of (+)-α-pinene by SnitCSP2, while for terpenes, ILE16, ALA25, TYR26, CYS29, GLU39, THR37, and GLY40 were vital for a stable binding system. We identified three potential ligands and analyzed the interaction patterns of the proteins with them to provide a favorable molecular basis for regulating insect behavioral interactions and developing new pest control strategies.

[Display omitted] • Odorant binding proteins and chemosensory proteins were identified in Protegira songi. • Putative ligand binding cavities were found in the predicted protein structures. • Sixteen PsonOBPs and eleven PsonCSPs were found in adult antennae or/and larval head. • PsonOBPs and PsonCSPs may play important roles in P. songi olfactory system. The larvae of Protegira songi Chen & Zhang are notorious defoliator specifically infesting the medicinal plant Eucommia ulmoides Oliver. In insects, Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) are highly expressed in olfactory organs and play essential roles in perceiving female pheromone and host volatiles by binding and transporting odorant compounds. In this study, 17 OBPs and 16 CSPs were identified from P. songi by transcriptome analysis. Sequence alignment indicated that PsonOBPs and PsonCSPs share no identity with each other in protein sequence. The overall structures of PsonOBPs and PsonCSPs are mainly constructed by α-helix bundles and loops. But the distribution of conserved cysteine residues and arrangement of α-helix are varied between PsonOBPs and PsonCSPs. Putative ligand binding cavities were found in these predicted protein structures. Expression profiles of PsonOBPs and PsonCSPs in adults and larvae were determined by RT-PCR which showed that 16 PsonOBPs and 11 PsonCSPs were expressed in adult antennae or/and larval head. PsonGOBPs, PsonPBPs, PsonOBP5, PsonOBP7, and PsonCSP6 were preferentially expressed in olfactory associated tissues. Our results suggest that the PsonOBPs and PsonCSPs that contain ligand cavity and are highly enriched in olfactory tissues may play roles in chemosensory perception of P. songi , and the transcriptome data may facilitate the studies on the olfactory mechanism of P. songi specifically choosing E. ulmoides as host. [ABSTRACT FROM AUTHOR]

In insects, chemosensory proteins (CSPs) play an important role in the perception of the external environment and have been widely used for protein-binding characterization. Riptortus pedestris has received increased attention as a potential cause of soybean staygreen syndrome in recent years. In this study, we found that RpedCSP4 expression in the antennae of adult R. pedestris increased with age, with no significant difference in expression level observed between males and females, as determined through quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, we investigated the ability of RpedCSP4 to bind various ligands (five aggregated pheromone components and 13 soybean volatiles) using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP4 binds to three aggregated pheromone components of R. pedestris, namely, ((E)-2-hexenyl (Z)-3-hexenoate (E2Z3), (E)-2-hexenyl (E)-2-hexenoate (E2E2), and (E)-2-hexenyl hexenoate (E2HH)), and that its binding capacities are most stable under acidic condition. Finally, the structure and protein-ligand interactions of RpedCSP4 were further analyzed via homology modeling, molecular docking, and targeted mutagenesis experiments. The L29A mutant exhibited a loss of binding ability to these three aggregated pheromone components. Our results show that the olfactory function of RpedCSP4 provides new insights into the binding mechanism of RpedCSPs to aggregation pheromones and contributes to discover new target candidates that will provide a theoretical basis for future population control of R. pedestris. [Display omitted] • The expression of RpedCSP4 in the adult antennae increased with age. • We acquired a high-quality recombinant RpedCSP4 protein. • RpedCSP4 had binding affinity to E2Z3, E2E2, and E2HH and most stable at pH 5. • L29A mutant should participate in the interaction between RpedCSP4 and ligands. [ABSTRACT FROM AUTHOR]

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

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

Abstract Background The blood-feeding behavior evolved multiple times in Insecta lineages and it represents an excellent opportunity to study patterns of convergent molecular evolution regarding this habit. In insects the expansion of some gene families is linked with blood-feeding behavior, but a wide study comparing the evolution of these gene families among different lineages is still missing. Here we gathered genomic data from six independently-evolved hematophagous lineages, aiming to identify convergent expansions and/or contractions of gene families in hematophagous lineages of insects. Results We found four rapidly evolving gene families shared by at least two hematophagous independently-evolved lineages, including a heat-shock and a chemosensory protein. On the expression of these four rapidly evolving gene families we found more genes expressed in mated individuals compared with virgin individuals in rapidly-expanded families and more genes expressed in non-blood-feeding individuals compared with blood-feeding individuals in rapidly-contracted families. Conclusion Our results reveal a new set of candidate genes to be explored in further analysis to help the development of new strategies to deal with blood-feeding vectors and also presents a new perspective to study the evolution of hematophagy identifying convergent molecular patterns.

Chemosensory proteins (CSPs) constitute a class of olfactory proteins localized in insect sensory organs that serve a crucial function in decoding external chemical stimuli. This study aims to elucidate the involvement of CrufCSP3 in olfactory perception within the context of Cotesia ruficrus , an indigenous endoparasitoid targeting the invasive pest Spodoptera frugiperda . Through fluorescence-competitive binding assays and site-directed mutagenesis, we pinpointed four amino acids as pivotal residues involved in the interaction between CrufCSP3 and five host-related compounds. Subsequent RNA interference experiments targeting CrufCSP3 unveiled a reduced sensitivity to specific host-related compounds and a decline in the parasitism rate of the FAW larvae. These findings unequivocally indicate the essential role of CrufCSP3 in the chemoreception process of C. ruficrus . Consequently, our study not only sheds light on the functional importance of CSPs in parasitic wasp behavior but also contributes to the development of eco-friendly and efficacious wasp behavior modifiers for effectively mitigating pest population surges.

Chemosensory proteins (CSPs) have been identified in the sensory tissues of various insect species and are believed to be involved in chemical communication in insects. However, the physiological roles of CSPs in Halyomorpha halys , a highly invasive insect species, are rarely reported. Here, we focused on one of the antennal CSPs (HhalCSP15) and determined whether it was involved in olfactory perception. Reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) analysis showed that HhalCSP15 was enriched in nymph and male and female adult antennae, indicating its possible involvement in the chemosensory process. Fluorescence competitive binding assays revealed that three of 43 natural compounds showed binding abilities with HhalCSP15, including β-ionone (Ki =11.9±0.6μM), cis -3-hexen-1-yl benzoate (Ki =10.5±0.4μM), and methyl (2 E ,4 E ,6 Z)-decatrienoate (EEZ -MDT; Ki =9.6±0.8μM). Docking analysis supported the experimental affinity for the three ligands. Additionally, the electrophysiological activities of the three ligands were further confirmed using electroantennography (EAG). EEZ -MDT is particularly interesting, as it serves as a kairomone when H. halys forages for host plants. We therefore conclude that HhalCSP15 might be involved in the detection of host-related volatiles. Our data provide a basis for further investigation of the physiological roles of CSPs in H. halys , and extend the olfactory function of CSPs in stink bugs. [ABSTRACT FROM AUTHOR]

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As potential molecular targets for developing novel pest management strategies, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) have been considered to initiate odor recognition in insects. Herein, we investigated the OBPs and CSPs in a major global crop pest (Spodoptera exempta). Using transcriptome analysis, we identified 40 OBPs and 33 CSPs in S. exempta, among which 35 OBPs and 29 CSPs had intact open reading frames. Sequence alignment indicated that 30 OBPs and 23 CSPs completely contained the conserved cysteines. OBPs of lepidopteran insects usually belonged to classical, minus-C, and plus-C groups. However, phylogenetic analyses indicated that we only identified 28 classical and seven minus-C OBPs in S. exempta, suggesting that we might have missed some typical OBPs in lepidopteran insects, probably due to their low expression levels. All of the CSPs from S. exempta clustered with the orthologs of other moths. The identification and expression of the OBPs and CSPs were well studied in insect adults by transcriptional analyses, and herein we used samples at different stages to determine the expression of OBPs and CSPs in S. exempta. Interestingly, our data indicated that several OBPs and CSPs were especially or more highly expressed in larvae or pupae than other stages, including three exclusively (SexeOBP13, SexeOBP16 and SexeCSP23) and six more highly (SexeOBP15, SexeOBP37, SexeCSP4, SexeCSP8, SexeCSP19, and SexeCSP33) expressed in larvae, two exclusively (SexeCSP6 and SexeCSP20) and three more highly (SexeOBP18, SexeCSP17, and SexeCSP26) expressed in pupae. Usually, OBPs and CSPs had both male- and female-biased expression patterns in adult antennae. However, our whole-body data indicated that all highly expressed OBPs and CSPs in adults were male-biased or did not differ, suggesting diverse OBP and CSP functions in insect adults. Besides identifying OBPs and CSPs as well as their expression patterns, these results provide a molecular basis to facilitate functional studies of OBPs and CSPs for exploring novel management strategies to control S. exempta. [ABSTRACT FROM AUTHOR]

The common cutworm, Spodoptera litura , is a polyandrous moth with high reproductive ability. Sexual reproduction is a unique strategy for survival and reproduction of population in this species. However, to date available information about its reproductive genes is rare. Here, we combined transcriptomics, genomics and proteomics approaches to characterize reproductive-related proteins in S. litura. Illumina sequencing in parallel with the reference genome led to the yields of 12,161 reproductive genes, representing 47.83% of genes annotated in the genome. Further, 524 genes of 19 specific gene families annotated in the genome were detected in reproductive tissues of both sexes, some of which exhibited sex-biased and/or tissue-enriched expression. Of these, manual efforts together with the transcriptome analyses re-annotated 54 odorant binding proteins (OBPs) and 23 chemosensory proteins (CSPs) with an increase of 18 OBPs and one CSP compared to those previously annotated in the genome. Interestingly, at least 35 OBPs and 22 CSPs were transcribed in at least one reproductive tissue, suggestive of their involvement in reproduction. Further proteomic analysis revealed 2381 common proteins between virgin and mated female reproductive systems, 79 of which were differentially expressed. More importantly, 74 proteins exclusive to mated females were identified as transferred relatives, coupled with their specific or high expression in male reproductive systems. Of the transferred proteins, several conserved protein classes across insects were observed including OBPs, serpins, trypsins and juvenile hormone-binding proteins. Our current study has extensively surveyed reproductive genes in S. litura with an emphasis on the roles of OBPs and CSPs in reproduction, and identifies potentially transferred proteins serving as modulators of female post-mating behaviors. [Display omitted] • Transcriptome of eight reproductive tissues of both sexes in Spodoptera litura are sequenced and characterized. • Integrated analysis leads to the yields of 54 OBPs and 23 CSPs with 19 novel candidates. • 35 OBPs and 22 CSPs are associated with reproduction. • LC-MS/MS analysis identifies 79 differentially expressed proteins between virgin and mating female reproductive systems. • Proteomic analysis identifies 74 proteins transferred from males to females during mating. [ABSTRACT FROM AUTHOR]

Chemoreception is of great importance for survival of insects. Chemosensory proteins (CSPs) are believed to be involved in the perireceptor events of chemosensory system in many insect species, but this has not been clarified in the citrus fruit fly, Bactrocera minax, a serious dipteran pest of citrus crops. Previous studies indicated that four CSP genes were identified in many fly speices in Diptera. In the present study, we also identified four CSP genes in B. minax, namely BminCSP1–4, from a transcriptome database. All CSP proteins encoded by these genes bear the typical hallmarks of the CSP family: an N‐terminal signal peptide and the four highly conserved cysteine residues. Phylogenetic analysis comparing with other dipteran CSPs indicated that dipteran CSPs may evolved from three ancestral CSP genes, and revealed the sequence diversities of BminCSPs and showed that BminCSP1–4 are clustered in separate groups, indicating the possibility of their contrasting function in B. minax. Furthermore, the tissue distribution of the four CSP genes in adult B. minax was analyzed by real‐time quantitiative PCR. The results demonstrated that BminCSP3 are significantly transcriptionally enriched in antennae; BminCSP4 expressed primarily in heads; BminCSP1 and BminCSP2 showed high expressions in different tissues, such as antennae, abdomens and wings. Based on these findings, the different implications for the functions of BminCSPs are discussed. This study will offer a significant indication for further functional studies of the CSPs in B. minax. [ABSTRACT FROM AUTHOR]

Chemosensory proteins (CSPs) have been identified in the sensory tissues of various insect species and are believed to be involved in chemical communication in insects. However, the physiological roles of CSPs in Halyomorpha halys, a highly invasive insect species, are rarely reported. Here, we focused on one of the antennal CSPs (HhalCSP15) and determined whether it was involved in olfactory perception. Reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) analysis showed that HhalCSP15 was enriched in nymph and male and female adult antennae, indicating its possible involvement in the chemosensory process. Fluorescence competitive binding assays revealed that three of 43 natural compounds showed binding abilities with HhalCSP15, including β-ionone (Ki=11.9±0.6μM), cis-3-hexen-1-yl benzoate (Ki=10.5±0.4μM), and methyl (2E,4E,6Z)-decatrienoate (EEZ-MDT; Ki=9.6±0.8μM). Docking analysis supported the experimental affinity for the three ligands. Additionally, the electrophysiological activities of the three ligands were further confirmed using electroantennography (EAG). EEZ-MDT is particularly interesting, as it serves as a kairomone when H. halys forages for host plants. We therefore conclude that HhalCSP15 might be involved in the detection of host-related volatiles. Our data provide a basis for further investigation of the physiological roles of CSPs in H. halys, and extend the olfactory function of CSPs in stink bugs.

Insects stimulate specific behaviors by correctly recognizing scent molecules in the external environment. Sirex nitobei, a wood-boring wasp species native to Asia with a distribution area that includes the Palaearctic and Oriental regions, is a significant pest of conifers. Focusing on the molecular mechanism of protein-ligand binding, this study resolved the tissue expression profile of CSP4 from S. nitobei (SnitCSP4) and probed its binding properties with target ligands using molecular docking and dynamics simulations to verify the odor recognition function of this protein. The open reading frame (ORF) of SnitCSP4 was 396 bp, encoding 131 amino acids. Tissue expression analysis revealed that SnitCSP4 was significantly expressed in female antennae and docking showed that all ligands were bound in hydrophobic cavities and close to many hydrophobic amino acid residues. GLN68 and LEU49 were important amino acid residues for SnitCSP4 to bind various odors, and THR9 was the key ligand-binding site in identifying (-)-globulol in the SnitCSP4. Molecular dynamics verified the docking results, confirming that SnitCSP4 bound well to two sex pheromone molecules, three host plant volatiles, and three symbiotic fungal volatiles, with (Z)-7-heptacosene, (Z)-9-nonacosene, and (-)-globulol binding being the most highly stable. These results mean that SnitCSP4 is critical for insects recognizing scent molecules, providing a favorable molecular basis for regulating the behavioral interactions between S. nitobei and the environment, and offering the possibility of developing new strategies for more environmentally friendly and effective control.

Unraveling the origin of molecular pathways underlying the evolution of adaptive traits is essential for understanding how new lineages emerge, including the relative contribution of conserved ancestral traits and newly evolved derived traits. Here, we investigated the evolutionary divergence of sex pheromone communication from moths (mostly nocturnal) to butterflies (mostly diurnal) that occurred ~119 million years ago. In moths, it is the females that typically emit pheromones to attract male mates, but in butterflies males emit pheromones that are used by females for mate choice. The molecular bases of sex pheromone communication are well understood in moths, but they have remained relatively unexplored in butterflies. We used a combination of transcriptomics, real time qPCR, and phylogenetics to identify genes involved in the different steps (i.e., production, regulation, and reception) of sex pheromone communication of the butterfly Bicyclus anynana. Our results show that the biosynthesis and reception of sex pheromones relies both on moth-specific gene families (reductases) and on more ancestral insect gene families (desaturases, olfactory receptors, odorant binding proteins). Interestingly, B. anynana appears to use what was believed to be the moth-specific neuropeptide Pheromone Biosynthesis Activating Neuropeptide (PBAN) for regulating sex pheromone production. Altogether, our results suggest that a mosaic pattern best explains how sex pheromone communication evolved in butterflies, with some molecular components derived from moths, and others conserved from more ancient insect ancestors. This is the first large-scale investigation of the genetic pathways underlying sex pheromone communication in a butterfly.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) play a fundamental role in insect olfaction. Galeruca daurica (Joannis) is a new pest with outbreak status in the Inner Mongolia grasslands, northern China. In this study, six olfactory protein genes (GdauOBP1, GdauOBP6, GdauOBP10, GdauOBP15, GdauCSP4, and GdauCSP5) were cloned by RACE and expressed by constructing a prokaryotic expression system. Their binding affinities to 13 compounds from host volatiles (Allium mongolicum) were determined by fluorescence-binding assay. In order to further explore the olfactory functions of GdauOBP15 and GdauCSP5, RNA interference (RNAi) and electroantennogram (EAG) experiments were conducted. Ligand-binding assays showed that the binding properties of the six recombinant proteins to the tested volatiles were different. GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 could bind several tested ligands of host plants. It was suspected that GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 were related to the host location in G. daurica. We also found that there were different EAG responses between males and females when the GdauOBP15 and GdauCSP5 genes were silenced by RNAi. The EAG response of G. daurica females to 2-hexenal was significantly decreased in dsRNA-OBP15-injected treatment compared to the control, and the dsRNA-CSP5-treated females significantly reduced EAG response to eight tested host volatiles (1,3-dithiane, 2-hexenal, methyl benzoate, dimethyl trisulfide, myrcene, hexanal, 1,3,5-cycloheptatriene, and p-xylene). However, the EAG response had no significant difference in males. Both GdauOBP15 and GdauCSP5 may have different functions between males and females in G. daurica and may play more important roles in females searching for host plants.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) play a fundamental role in insect olfaction. Galeruca daurica (Joannis) is a new pest with outbreak status in the Inner Mongolia grasslands, northern China. In this study, six olfactory protein genes (GdauOBP1 , GdauOBP6 , GdauOBP10 , GdauOBP15 , GdauCSP4 , and GdauCSP5) were cloned by RACE and expressed by constructing a prokaryotic expression system. Their binding affinities to 13 compounds from host volatiles (Allium mongolicum) were determined by fluorescence-binding assay. In order to further explore the olfactory functions of GdauOBP15 and GdauCSP5 , RNA interference (RNAi) and electroantennogram (EAG) experiments were conducted. Ligand-binding assays showed that the binding properties of the six recombinant proteins to the tested volatiles were different. GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 could bind several tested ligands of host plants. It was suspected that GdauOBP6, GdauOBP15, GdauCSP4, and GdauCSP5 were related to the host location in G. daurica. We also found that there were different EAG responses between males and females when the GdauOBP15 and GdauCSP5 genes were silenced by RNAi. The EAG response of G. daurica females to 2-hexenal was significantly decreased in dsRNA-OBP15-injected treatment compared to the control, and the dsRNA-CSP5-treated females significantly reduced EAG response to eight tested host volatiles (1,3-dithiane, 2-hexenal, methyl benzoate, dimethyl trisulfide, myrcene, hexanal, 1,3,5-cycloheptatriene, and p-xylene). However, the EAG response had no significant difference in males. Both GdauOBP15 and GdauCSP5 may have different functions between males and females in G. daurica and may play more important roles in females searching for host plants. [ABSTRACT FROM AUTHOR]

Antennae and legs (primarily the tarsal segments) of insects are the foremost sensory organs that contact a diverse range of toxic chemicals including insecticides. Binding proteins expressed in the two tissues are potential molecular candidates serving as the binding and sequestering of insecticides, like chemosensory proteins (CSPs). Insect CSPs endowed with multiple roles have been suggested to participate in insecticide resistance, focusing mainly on moths, aphids and mosquitos. Yet, the molecular underpinnings underlying the interactions of cerambycid CSPs and insecticides remain unexplored. Here, we present binding properties of three antenna- and tarsus-enriched RhorCSPs (RhorCSP1, CSP2 and CSP3) in Rhaphuma horsfieldi to eight insecticide classes totaling 15 chemicals. From the transcriptome of this beetle, totally 16 CSP-coding genes were found, with seven full-length sequences. In phylogeny, these RhorCSPs were distributed dispersedly in different clades. Expression profiles revealed the abundant expression of RhorCSP1 , CSP2 and CSP3 in antennae and tarsi, thus as representatives for studying the protein-insecticide interactions. Binding assays showed that the three RhorCSPs were tuned differentially to insecticides but exhibited the highest affinities with hexaflumuron, chlorpyrifos and rotenone (dissociation constants <13 μM). In particular, RhorCSP3 could interact strongly with 10 of tested insecticides, of which four residues (Tyr25, Phe42, Val65 and Phe68) contributed significantly to the binding of six, four, three and four ligands, respectively. Of these, the binding of four mutated RhorCSP3s to a botanical insecticide rotenone was significantly weakened compared to the wildtype protein. Furthermore, we also evidenced that RhorCSP3 was a broadly-tuned carrier protein in response to a wide variety of plant odorants outside insecticides. Altogether, our findings shed light on different binding mechanisms and odorant-tuning profiles of three RhorCSPs in R. horsfieldi and identify key residues of the RhorCSP3-insecticide interactions. [Display omitted] • RhorCSP1 , CSP2 and CSP3 genes in Rhaphuma horsfieldi were highly enriched in antennae and tarsi. • Three RhorCSPs exhibited different odorant-tuning profiles and binding abilities to insecticides. • Residues Tyr25, Phe42, Val65 and Phe68 significantly weakened the binding of RhorCSP3 to some insecticides. • RhorCSP3 could be tuned broadly to various insecticides and plant odorants. [ABSTRACT FROM AUTHOR]

Abstract Background Olfaction and gustation underlie behaviors that are crucial for insect fitness, such as host and mate selection. The detection of semiochemicals is mediated via proteins from large and rapidly evolving chemosensory gene families; however, the links between a species’ ecology and the diversification of these genes remain poorly understood. Hence, we annotated the chemosensory genes from genomes of select wood-boring coleopterans, and compared the gene repertoires from stenophagous species with those from polyphagous species. Results We annotated 86 odorant receptors (ORs), 60 gustatory receptors (GRs), 57 ionotropic receptors (IRs), 4 sensory neuron membrane proteins (SNMPs), 36 odorant binding proteins (OBPs), and 11 chemosensory proteins (CSPs) in the mountain pine beetle (Dendroctonus ponderosae), and 47 ORs, 30 GRs, 31 IRs, 4 SNMPs, 12 OBPs, and 14 CSPs in the emerald ash borer (Agrilus planipennis). Four SNMPs and 17 CSPs were annotated in the polyphagous wood-borer Anoplophora glabripennis. The gene repertoires in the stenophagous D. ponderosae and A. planipennis are reduced compared with those in the polyphagous A. glabripennis and T. castaneum, which is largely manifested through small gene lineage expansions and entire lineage losses. Alternative splicing of GR genes was limited in D. ponderosae and apparently absent in A. planipennis, which also seems to have lost one carbon dioxide receptor (GR1). A. planipennis has two SNMPs, which are related to SNMP3 in T. castaneum. D. ponderosae has two alternatively spliced OBP genes, a novel OBP “tetramer”, and as many as eleven IR75 members. Simple orthology was generally rare in beetles; however, we found one clade with orthologues of putative bitter-taste GRs (named the “GR215 clade”), and conservation of IR60a from Drosophila melanogaster. Conclusions Our genome annotations represent important quantitative and qualitative improvements of the original datasets derived from transcriptomes of D. ponderosae and A. planipennis, facilitating evolutionary analysis of chemosensory genes in the Coleoptera where only a few genomes were previously annotated. Our analysis suggests a correlation between chemosensory gene content and host specificity in beetles. Future studies should include additional species to consolidate this correlation, and functionally characterize identified proteins as an important step towards improved control of these pests.

Social insects have evolved acute mechanisms for sensing and mitigating the spread of microbial pathogens within their communities that include complex behaviors such as grooming and sanitation. Chemical sensing involves detection and transport of olfactory and other chemicals that are mediated by at least two distinct classes of small molecular weight soluble proteins known as chemosensory- and odorant binding proteins (CSPs and OBPs, respectively) that exist as protein families in all insects. However, to date, a systematic examination of the expression of these genes involved in olfactory and other pathways to microbial infection has yet to be reported. The red imported fire ant, Solenopsis invicta, is one of the most successful invasive organisms on our planet. Here, we examined the temporal gene expression profiles of a suite of S. invicta CSPs (SiCSPs1-22) and OBPs (SiOBPs1-16) in response to infection by the broad host range fungal insect pathogen, Beauveria bassiana. Our data show that within 24 h post-infection, i.e., before the fungus has penetrated the host cuticle, the expression of SiCSPs and SiOBPs is altered (mainly increased compared to uninfected controls), followed by suppression of SiCSP and select SiOBP expression 48 h post-infection and mixed responses at 72 h post-infection. A smaller group of SiBOPs, however, appeared to respond to fungal infection, with expression of SiOBP15 consistently higher during fungal infection over the time course examined. These data indicate dynamic gene expression responses of CSPs and OBPs to fungal infection that provide clues to mechanisms that might mediate detection of microbial pathogens, triggering grooming, and nest sanitation.

Social insects have evolved acute mechanisms for sensing and mitigating the spread of microbial pathogens within their communities that include complex behaviors such as grooming and sanitation. Chemical sensing involves detection and transport of olfactory and other chemicals that are mediated by at least two distinct classes of small molecular weight soluble proteins known as chemosensory- and odorant binding proteins (CSPs and OBPs, respectively) that exist as protein families in all insects. However, to date, a systematic examination of the expression of these genes involved in olfactory and other pathways to microbial infection has yet to be reported. The red imported fire ant, Solenopsis invicta , is one of the most successful invasive organisms on our planet. Here, we examined the temporal gene expression profiles of a suite of S. invicta CSPs (SiCSPs1-22) and OBPs (SiOBPs1-16) in response to infection by the broad host range fungal insect pathogen, Beauveria bassiana. Our data show that within 24 h post-infection, i.e., before the fungus has penetrated the host cuticle, the expression of SiCSPs and SiOBPs is altered (mainly increased compared to uninfected controls), followed by suppression of SiCSP and select SiOBP expression 48 h post-infection and mixed responses at 72 h post-infection. A smaller group of SiBOPs , however, appeared to respond to fungal infection, with expression of SiOBP15 consistently higher during fungal infection over the time course examined. These data indicate dynamic gene expression responses of CSPs and OBPs to fungal infection that provide clues to mechanisms that might mediate detection of microbial pathogens, triggering grooming, and nest sanitation. [ABSTRACT FROM AUTHOR]

Chemosensory proteins (CSPs) are a class of transporters in arthropods. Deeper research on CSPs showed that CSPs may be involved in some physiological processes beyond chemoreception, such as insect resistance to pesticides. We identified two upregulated CSPs in two resistant strains of Aphis gossypii Glover. To understand their role in the resistance of aphids to pesticides, we performed the functional verification of CSP1 and CSP4 in vivo and in vitro. Results showed that the sensitivity of the thiamethoxam-resistant strain to thiamethoxam increased significantly with the silencing of CSP1 and CSP4 by RNAi (RNA interference), and the sensitivity of the spirotetramat-resistant strain to spirotetramat increased significantly with the silencing of CSP4. Transgenic Drosophila melanogaster expressing CSPs exhibited stronger resistance to thiamethoxam, spirotetramat, and alpha-cypermethrin than the control did. In the bioassay of transgenic Drosophila, CSPs showed different tolerance mechanisms for different pesticides, and the overexpressed CSPs may play a role in processes other than resistance to pesticides. In brief, the present results prove that CSPs are related to the resistance of cotton aphids to insecticides.

Ophraella communa is an efficient biocontrol agent used against the invasive weed Ambrosia artemisiifolia. It is an herbivorous insect that feeds on specific plants; the olfactory functions of this insects plays an important role in their search for host plants. There are no reports on O. communa sensilla types, morphology, or chemosensory protein (CSP) genes. In this study, we observed the external structure and distribution of antennal sensilla in adult O. communa antennae by scanning electron microscopy; moreover, we cloned 11 CSPs (CSP1–CSP11) and elucidated their tissue-expression profiles using quantitative real-time polymerase chain reaction. Six types of sensilla were identified: sensilla trichodea (including two subtypes), sensilla chaetica, sensilla basiconica (including two subtypes), sensilla styloconica, sensilla coeloconica, and Böhm bristles. Both male and female antennae had all six types of sensilla, and no sexual dimorphism was noted in sensillar types or distribution. We also found that the expression levels of CSP2, CSP3, CSP4, CSP6, and CSP7 in male and female antennae were higher than those in other tissues, which suggests that these five CSPs may be related to olfactory function in O. communa. Ultimately, our results lay the foundation for interpreting the olfactory functions of adult O. communa.

Olfaction plays indispensable roles in insect behavior such as host location, foraging, oviposition, and avoiding predators. Chemosensory proteins (CSPs) can discriminate the hydrophobic odorants and transfer them to the odorant receptors. Presently, CSPs have been identified in many insect species. However, their presence and functions remain unknown in Bactrocera dorsalis , a destructive and invasive insect pest in the fruit and vegetable industry. Here, we annotated eight CSP genes in the genome of B. dorsalis . The results of quantitative real-time polymerase chain reaction (RT-qPCR) showed that BdorCSP3 was highly expressed in the antennae. Molecular docking and in vitro binding assays showed that BdorCSP3 had a good binding ability to host volatiles methyl eugenol (ME, male-specific attractant) and β-caryophyllene (potential female attractant). Subsequently, CRISPR/Cas9 was used to generate BdorCSP3 -/- mutants. Electroantennograms (EAGs) and behavioral assays revealed that male mutants significantly reduced the preference for ME, while female mutants lost their oviposition preference to β-caryophyllene. Our data indicated that BdorCSP3 played important roles in the perception of ME and β-caryophyllene. The results not only expanded our knowledge of the olfaction perception mechanism of insect CSPs but also provided a potential molecular target for the control of B. dorsalis .

The brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), is a serious agricultural and urban pest that has become an invasive species in many parts of the world. Olfaction plays an indispensable role in regulating insect behaviors, such as host plant location, partners searching, and avoidance of predators. In this study, we sequenced and analyzed the antennal transcriptomes of both male and female adults of H. halys to better understand the olfactory mechanisms in this species. A total of 241 candidate chemosensory genes were identified, including 138 odorant receptors (ORs), 24 ionotropic receptors (IRs), 15 gustatory receptors (GRs), 44 odorant-binding proteins (OBPs), 17 chemosensory proteins (CSPs), and three sensory neuron membrane proteins (SNMPs). The results of semi-quantitative reverse transcription PCR (RT-PCR) assays showed that some HhalOBP and HhalCSP genes have tissue-specific and sex-biased expression patterns. Our results provide an insight into the molecular mechanisms of the olfactory system in H. halys and identify potential novel targets for pest control strategies.

Chemosensory proteins (CSPs) are a family of small, soluble proteins that play a crucial role in transporting odorant and pheromone molecules in the insect chemosensory system. Recent studies reveal that they also function in development, nutrient metabolism and insecticide resistance. In-depth and systematic characterization of previously unknown CSPs will be valuable to investigate more detailed functionalities of this protein family. Here, we identified 27 CSP genes from the genome and transcriptome sequences of cotton bollworm, Helicoverpa armigera (Hübner). The expression patterns of these genes were studied by using transcriptomic data obtained from different tissues and stages. The results demonstrate that H. armigera CSP genes are not only highly expressed in chemosensory tissues, such as antennae, mouthparts, and tarsi, but also in the salivary glands, cuticle epidermis, and hind gut. HarmCSP6 and 22 were selected as candidate CSPs for expression in Escherichia coli and purification. A new method was developed that significantly increased the HarmCSP6 and 22 expression levels as soluble recombinant proteins for purification. This study advances our understanding of insect CSPs and provides a new approach to highly express recombinant CSPs in E. coli.

• A total of 21 CSP genes were identified in Pieris rapae. • P. rapae CSP genes were tandemly arrayed in the genome scaffold. • Four genes were highly expressed in the antennae. • This is the first study of large-scale discovery for CSP s in small white butterfly. Chemosensory proteins (CSPs) play a crucial role in olfactory recognition in insects. The small white butterfly Pieris rapae —a major pest of Brassicaceae vegetables, which causes enormous economic losses—uses olfaction to locate its host plants. However, the molecular mechanism of olfaction in this species remains unknown. Herein, we performed a genome-wide and transcriptome-wide analysis of CSP genes in P. rapae and identified 21 CSP s (PrapCSP1 to PrapCSP21). Proteins encoded by these genes showed typical characteristics of CSPs—an N-terminal signal peptide and four positionally conserved cysteine residues. BLASTX analysis indicated that most P. rapae CSPs showed high amino acid identity with their respective orthologs in other lepidopterans. Phylogenetic analysis showed that most P. rapae CSPs were well segregated and were clustered into different branches. The 21 genes were located on six genomic scaffolds, and most genes were tandemly arrayed. Quantitative reverse transcription-PCR showed that PrapCSP3 , 4, 16 and 21 had the highest expression level in the antennae; PrapCSP7 and PrapCSP18 were mainly expressed in the ovaries, and PrapCSP9 and PrapCSP17 were leg-enriched. PrapCSP11 and PrapCSP20 were found mainly in the heads and testes, respectively. Our findings provide a solid foundation for studying the function of these genes. [ABSTRACT FROM AUTHOR]