Chemosensory Proteins Research Articles

Decoding the genomic terrain: functional insights into 14 chemosensory proteins in whitefly Bemisia tabaci Asia II-1

Genome-wide analysis of Bemisia tabaci Asia II-1 unravelled for the first-time full-length sequences of 14 chemosensory proteins (CSPs), their exon-intron boundaries, insertion sites of retrotransposons, and clustering patterns on chromosomes. All the CSPs sans CSP6 have an N-terminal signal peptide. The presence of OS-D superfamily and PhBP domains in different CSPs suggests their roles in chemosensory signal transduction and pheromone binding. Motif analysis reveals the conservation and cohesiveness of CSPs in hemiptera. The phylogenetic analysis uncovers the evolutionary lineages of Hemipteran CSPs. RT-qPCR analysis showed spatial expression of CSPs in different body tissues of B. tabaci adults. In-silico docking analysis showed high-affinity binding of CSP 1 and 5 with two insecticides, imidacloprid and fipronil, with energy values ranging from − 5.8 to -9.3 kcal/mol, along with the details of interacting aminoacidic residues in the hydrophobic binding pockets of these two CSPs. Further functional validation was done through insecticide bioassays and RNAi. This study provides novel insights into the genomic architecture of CSPs in B. tabaci Asia II-1, and functional characterisation suggests that CSP1 and 5 genes may have indirect roles in insecticide resistance. It lays the foundation for further research on developing new control strategies for B. tabaci.

A larval expressed chemosensory protein involved in recognition of anthocyanins in Spodoptera litura (Lepidoptera: Noctuidae).

Anthocyanins are secondary metabolites which act as diverse functions during plant growth. Insects can discriminate host plants by their sensitive gustatory systems. It is hypothetical that chemosensory proteins (CSPs) play a crucial role in regulating this behavioral process. However, the underlying molecular mechanisms remain obscure. In the present study, we characterized a CSP SlitCSP8 from the Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). Quantitative real-time-polymerase chain reaction analysis demonstrated that SlitCSP8 was mainly expressed in the head of the 7th S. litura larvae, especially labrum. Further, recombinant SlitCSP8 was obtained using bacterial expression system. Fluorescence competitive binding assays demonstrated that the purified SlitCSP8 exhibited a strong binding affinity to anthocyanins, a natural compound derived from the host plant. Silencing SlitCSP8 through RNAi significantly reduced the sensitivity of S. litura larvae to anthocyanins-treated leaf disks, the development from larva to pupae was not affected. These data provide insight into the molecular basis that CSP8 can detect anthocyanins in host plants by chemosensory system of insects. It can be further used in designing novel optimal food attractant targeting to the CSPs for pest control.

Recognition of a salivary effector by the TNL protein RCSP promotes effector-triggered immunity and systemic resistance in Nicotiana benthamiana.

Insects secret chemosensory proteins (CSPs) into plant cells as potential effector proteins during feeding. The molecular mechanisms underlying how CSPs activate plant immunity remain largely unknown. We show that CSPs from six distinct insect orders induce dwarfism when overexpressed in Nicotiana benthamiana. Agrobacterium-mediated transient expression of Nilaparvata lugens CSP11 (NlCSP11) triggered cell death and plant dwarfism, both of which were dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), neuregulin 1 (NRG1) and SENESCENCE-ASSOCIATED GENE 101 (SAG101), indicating the activation of effector-triggered immunity (ETI) in N. benthamiana. Overexpression of NlCSP11 led to stronger systemic resistance against Pseudomonas syringae DC3000 lacking effector HopQ1-1 and tobacco mosaic virus, and induced higher accumulation of salicylic acid (SA) in uninfiltrated leaves compared to another effector XopQ that is recognized by a Toll-interleukin-1 receptor (TIR) domain nucleotide-binding leucine-rich repeat receptor (TNL) called ROQ1 in N. benthamiana. Consistently, NlCSP11-induced dwarfism and systemic resistance, but not cell death, were abolished in N. benthamiana transgenic line expressing the SA-degrading enzyme NahG. Through large-scale virus-induced gene silencing screening, we identified a TNL protein that mediates the recognition of CSPs (RCSP), including aphid effector MP10 that triggers resistance against aphids in N. benthamiana. Co-immunoprecipitation, bimolecular fluorescence complementation and AlphaFold2 prediction unveiled an interaction between NlCSP11 and RCSP. Interestingly, RCSP does not contain the conserved catalytic glutamic acid in the TIR domain, which is required for TNL function. Our findings point to enhanced ETI and systemic resistance by a TNL protein via hyperactivation of the SA pathway. Moreover, RCSP is the first TNL identified to recognize an insect effector.

Antennal Transcriptome Screening and Identification of Chemosensory Proteins in the Double-Spine European Spruce Bark Beetle, Ips duplicatus (Coleoptera: Scolytinae).

The northern bark beetle, Ips duplicatus, is an emerging economic pest, reportedly infesting various species of spruce (Picea spp.), pine (Pinus spp.), and larch (Larix spp.) in Central Europe. Recent climate changes and inconsistent forest management practices have led to the rapid spread of this species, leaving the current monitoring strategies inefficient. As understanding the molecular components of pheromone detection is key to developing novel control strategies, we generated antennal transcriptomes from males and females of this species and annotated the chemosensory proteins. We identified putative candidates for 69 odorant receptors (ORs), 50 ionotropic receptors (IRs), 25 gustatory receptors (GRs), 27 odorant-binding proteins (OBPs), including a tetramer-OBP, 9 chemosensory proteins (CSPs), and 6 sensory neuron membrane proteins (SNMPs). However, no sex-specific chemosensory genes were detected. The phylogenetic analysis revealed conserved orthology in bark beetle chemosensory proteins, especially with a major forest pest and co-habitant, Ips typographus. Recent large-scale functional studies in I. typographus chemoreceptors add greater significance to the orthologous sequences reported here. Nevertheless, identifying chemosensory genes in I. duplicatus is valuable to understanding the chemosensory system and its evolution in bark beetles (Coleoptera) and, generally, insects.

Molecular Characterization of Odorant-Binding Protein Genes Associated with Host-Seeking Behavior in Oides leucomelaena.

The identification of odorant-binding proteins (OBPs) involved in host location by Oides leucomelaena (O. leucomelaena Weise, 1922, Coleoptera, Galerucinae) is significant for its biological control. Tools in the NCBI database were used to compare and analyze the transcriptome sequences of O. leucomelaena with OBP and other chemosensory-related proteins of other Coleoptera insects. Subsequently, MEGA7 was utilized for OBP sequence alignment and the construction of a phylogenetic tree, combined with expression profiling to screen for candidate antennae-specific OBPs. In addition, fumigation experiments with star anise volatiles were conducted to assess the antennae specificity of the candidate OBPs. Finally, molecular docking was employed to speculate on the binding potential of antennae-specific OBPs with star anise volatiles. The study identified 42 candidate OBPs, 8 chemosensory proteins and 27 receptors. OleuOBP3, OleuOBP5, and OleuOBP6 were identified as classic OBP family members specific to the antennae, which was confirmed by volatile fumigation experiments. Molecular docking ultimately clarified that OleuOBP3, OleuOBP5, and OleuOBP6 all exhibit a high affinity for β-caryophyllene among the star anise volatiles. We successfully obtained three antennae-specific OBPs from O. leucomelaena and determined their high-affinity volatiles, providing a theoretical basis for the development of attractants in subsequent stages.

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

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

Sex Differences in Antennal Transcriptome of Hyphantria cunea and Analysis of Odorant Receptor Expression Profiles.

Insects rely on olfaction for mating, finding oviposition sites, and locating hosts. Hyphantria cunea is a serious pest that severely damages forests. Differential expression analysis of olfactory-related genes between males and females is the basis for elucidating the functions of olfactory-related proteins in H. cunea. In this study, Illumina HiSeqTM 4000 high-throughput sequencing technology was used to perform transcriptome sequencing of the antennal tissues of adult male and female H. cunea. Functional annotation was conducted using the NR, Swiss-Prot, KOG, KEGG, and GO databases, and the results showed that the antennal transcriptome of adult H. cunea contained 50,158 unigenes. Differential expression analysis identified 3923 genes that were significantly differentially expressed between male and female antennae. A total of 221 olfactory-related genes were annotated, and 96 sex-biased genes were identified, including 13 odorant receptors (ORs), 48 odorant binding proteins (OBPs), 7 chemosensory proteins (CSPs), 10 ionotropic receptors (IRs), 10 sensory neuron membrane proteins (SNMPs), 2 gustatory receptors (GRs), and 6 odorant-degrading enzymes (ODEs), indicating that there were differences in olfaction between male and female H. cunea. Quantitative real-time PCR was used to verify the expression levels of 21 putative general odorant receptor genes in male and female antennae. HcunOR4 and HcunOR5 showed female-biased expression; HcunOR48, HcunOR49 and HcunOR50 showed male-biased expression. The results were consistent with the transcriptome differential analysis. The screening of male-biased odorant receptor genes might provide a theoretical basis for the functional characterization of odorant receptors for recognizing sex pheromones in H. cunea.

Disrupting the olfactory response in Monochamus saltuarius: Two potential target genes for controlling the vector insect of pine wilt disease.

Worldwide, pine forests have been threatened by a devastating pine wood disease caused by Bursaphelenchus xylophilus, with Monochamus saltuarius being a newly recorded vector of the disease in Northeast China. The olfactory system plays important roles in both feeding and oviposition during the adult stage of M.saltuarius, and olfactory gene function research is essential for gaining an understanding of the olfactory mechanisms of this pest. However, there is limited information available regarding olfactory gene functions in this pest. In the present study, we selected 7 olfactory genes, including 2 chemosensory proteins, 2 odorant-binding proteins, the odorant co-receptor and 2 odorant receptors, which were relatively highly expressed during the adult stage. We silenced these genes by RNA interference (RNAi), and real-time quantitative PCR was used to detect their expression levels after double-stranded RNA (dsRNA) injection. The results indicate that these genes were significantly downregulated at 2d post-dsRNA injection, and this was sustained until 5d post-injection. Electroantennography tests indicated that the knockdown of MsalOBP14 and MsalOrco impaired the olfactory response of M.saltuarius to 11 host volatiles and 1 sex pheromone compound. Y-tube experiments further confirmed that downregulated MsalOBP14 and MsalOrco expression led to olfactory dysfunction in M.saltuarius, which significantly lost selectivity. The results indicate that MsalOBP14 and MsalOrco play critical roles in sex communication and host volatile detection in M.saltuarius, and possibly represent 2 effective targets for controlling this forest pest through olfactory disruption.

Binding characteristics and structural dynamics of two general odorant-binding proteins with plant volatiles in the olfactory recognition of Glyphodes pyloalis

Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is the most destructive pest, causing severe damage to mulberry production in China's sericulture industry. The insecticide application in mulberry orchards poses a significant risk of poisoning to Bombyx mori. Shifting from insecticides to odor attractants is a beneficial alternative, but not much data is available on the olfactory system of G. pyloalis. We identified 114 chemosensory genes from the antennal transcriptome database of G. pyloalis, with 18 odorant-binding protein (OBP) and 17 chemosensory protein (CSP) genes significantly expressed in the antennae. Ligand-binding assays for two antennae-biased expressed general odorant-binding proteins (GOBPs) showed high binding affinities of GOBP1 to hexadecanal, β-ionone, and 2-ethylhexyl acrylate, while GOBP2 exhibited binding to 4-tert-octylphenol, benzyl benzoate, β-ionone, and farnesol. Computational simulations indicated that van der Waal forces predominantly contributed to the binding free energy in the binding processes of complexes. Among them, Phe12 of GOBP1 and Phe19 of GOBP2 were demonstrated to play crucial roles in their bindings to plant volatiles using site-directed mutagenesis experiments. Moreover, hexadecanal and β-ionone attracted G. pyloalis male moths in the behavioral assays, while none of the candidate plant volatiles significantly affected female moths. Our findings provide a comprehensive understanding of the molecular mechanisms underlying olfactory recognition in G. pyloalis, setting the groundwork for novel mulberry pests control strategies based on insect olfaction.

Identification and expression profiles of olfactory-related genes in the antennal transcriptome of Graphosoma rubrolineatum (Hemiptera: Pentatomidae).

Graphosoma rubrolineatum (Hemiptera: Pentatomidae) is an important pest of vegetables and herbs (e.g., Umbelliferae and Cruciferae) in China, Siberia, Korea, and Japan. Insects are highly dependent on their olfactory system to detect odorants. However, no molecular-mediated olfactory genes in G. rubrolineatum have yet been identified. In this study, we first established the antennal transcriptome of G. rubrolineatum and identified 189 candidate olfactory genes, including 31 odorant-binding proteins (OBPs), 15 chemosensory proteins (CSPs), four sensory neuron membrane proteins (SNMPs),94 odorant receptors (ORs), 23 ionotropic receptors (IRs), and 22 gustatory receptors (GRs). Additionally, phylogenetic trees were constructed for olfactory genes between G. rubrolineatum and other hemipteran insects. We also detected the expression profiles of ten OBPs, five CSPs, two SNMPs, five ORs, four IRs, and four GRs by real-time quantitative PCR. The results revealed that most genes (GrubOBP1/11/31, GrubCSP3/8, GrubSNMP1a/1b, GrubOrco/OR9/11/13, GrubGR1/4/22, GrubIR25/75h/76b/GluR1) were highly expressed in the antennae, GrubOBP13/31 and GrubCSP4/11/12 were highly expressed in the legs, while GrubOBP20 and GrubGR19 were highly expressed in the wings. Our results will enrich the gene inventory of G. rubrolineatum and provide further insight into the molecular chemosensory mechanisms of G. rubrolineatum.

In silico prediction of effector proteins in oil palm pest, Metisa plana

Metisa plana is one of the main oil palm leaf defoliators in Malaysia. Infestation of M. plana at the oil palm plantation caused severe yield loss in the past. Many control methods have been introduced to combat the infestation, but the problem persists till today. More information regarding the bagworm is needed to understand the pest-host interaction so that an effective control method can be properly designed. This study was carried out to predict the repertoire of effector proteins secreted by M. plana that could potentially suppress the host defence mechanisms. In silico screening of the secretory proteins from predicted genes was done by using several tools namely SignalP, TMHMM, TargetP and WOLFPSORT. Then, the acquired dataset was subjected to functional annotation through a similarity search against UniProt-Trembl database, gene ontology (GO) using BLAST2GO, InterPro protein domain classification, cluster of orthologous groups (COG), and KEGG pathway prediction. To determine the expression of the predicted genes, TPM value was calculated using the available transcriptomic dataset of M. plana. We identified a total of 948 secretory proteins from the initial screening of the 28,738 proteins predicted from BRAKER2 pipeline. Several proteins were classified as specific protein classes associated with effector functions in insect pests, namely detoxifying enzymes, lipases, peroxidases, Ca2+ binding proteins, and chemosensory and odorant binding proteins. This predicted effector data can be used as a reference for future works in validating the protein’s interaction and mechanisms with the oil palm defence responses.

Elucidation of ejaculatory bulb proteins in Bemisia tabaci Asia-1 and Asia II-1 and confirmation of their mating transfer via RNAi.

Bemisia tabaci, a significant agricultural pest in Asia, contains distinct genetic groups, Asia-1 and Asia II-1. Understanding its reproductive biology, particularly the role of ejaculatory bulb proteins (EBPs) in mating, is crucial. However, EBPs in B. tabaci were not well characterised until this study. The EBPs have been characterised in the Asia-1 and Asia II-1 genetic groups of the whitefly B. tabaci, prevalent in Asia. The transcriptomic analysis yielded over 40,000,000 and 30,000,000 annotated transcripts, respectively, from Asia II-1 and Asia-1. Differential gene expression revealed the presence of 270 upregulated and 198 downregulated genes, with significant differences between these two genetic groups. Orphan genes (1992 numbers) were identified in both genetic groups. We report, for the first time, full-length sequences of EBP genes from B. tabaci. The 10 EBPs each deduced in B. tabaci Asia-1 and Asia II-1 are structurally akin to chemosensory proteins having four conserved cysteine residues. Additionally, we did domain analysis, protein structure prediction, mapping of these EBPs in the chromosomes of B. tabaci, and phylogenetic analysis to track their evolutionary lineage. We have specifically demonstrated the transfer of EBPs from males to females during mating using qPCR and further validated the transfer of EBPs through RNAi. Specifically, we targeted the highly expressed EBPs (EBP-3, 7, and 8 in BtAsia1; EBP-8, 9, and 10 in BtAsia II-1) through feeding bioassays of dsRNAs. Tracking by qPCR revealed that the females, when mated with dsRNA-treated males, did not show expression of the specific EBP, suggesting that the silencing of these genes in males hinders the transfer of EBP to females during mating. Our findings provide novel insights into the genomic contours of EBPs in B. tabaci and underscore the potential of RNAi-based strategies for pest management by disrupting the reproductive processes.

Identification and characterization of olfactory gene families in Macrobrachium rosenbergii based on full-length transcripts and genome sequences

The olfactory gene families include odorant binding proteins (OBPs), chemosensory proteins (CSPs), olfactory receptors (ORs), ionotropic receptors (IRs) and gustatory receptors (GRs). To investigate the molecular function of olfactory perception in Macrobrachium rosenbergii, we integrated the full-length transcripts and whole-genome sequences to identify the olfactory gene families. In this study, a total of 38,955 full-length transcripts with an N50 length of 3383 bp were obtained through PacBio SMRT sequencing. Through the annotation of full-length transcripts and whole-genome sequences, several olfactory gene families were identified, including 18 MrORs, 16 MrIRs, 151 MrIGluRs (ionotropic glutamate receptors), 2 MrVIGluRs (variant ionotropic glutamate receptors) and 3 MrCRs (chemosensory receptors). Notably, the CRs were first identified in prawns and shrimps. Additionally, the olfactory gene families in M. nipponense were identified, comprising 4 MnORs, 21 MnIRs, 79 MnIGluRs, 5 MnVIGluRs, 1 MnGR and 1 MnOBP, using the available whole-genome sequences. Meanwhile, the external morphology of the chemical sensory organs of M. rosenbergii was explored, and the presence of plumose setae (PS), hard thorn setae (HTS), bamboo shoot setae (BSS), soft thorn setae (STS) and aesthetascs (AE) on the antennules, HTS and BSS on the second antennae, and PS on the pereiopods were observed by scanning electron microscope. This study provides valuable insights for future functional studies into the olfactory perception of crustaceans and establishes a theoretical basis for molecular design breeding in M. rosenbergii.

Antennal transcriptome analysis reveals sensory receptors potentially associated with host detection in the livestock pest Lucilia cuprina

BackgroundLucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae) is the main causative agent of flystrike of sheep in Australia and New Zealand. Female flies lay eggs in an open wound or natural orifice, and the developing larvae eat the host’s tissues, a condition called myiasis. To improve our understanding of host-seeking behavior, we quantified gene expression in male and female antennae based on their behavior.MethodsA spatial olfactometer was used to evaluate the olfactory response of L. cuprina mated males and gravid females to fresh or rotting beef. Antennal RNA-Seq analysis was used to identify sensory receptors differentially expressed between groups.ResultsLucilia cuprina females were more attracted to rotten compared to fresh beef (> fivefold increase). However, males and some females did not respond to either type of beef. RNA-Seq analysis was performed on antennae dissected from attracted females, non-attracted females and males. Transcripts encoding sensory receptors from 11 gene families were identified above a threshold (≥ 5 transcript per million) including 49 ATP-binding cassette transporters (ABCs), two ammonium transporters (AMTs), 37 odorant receptors (ORs), 16 ionotropic receptors (IRs), 5 gustatory receptors (GRs), 22 odorant-binding proteins (OBPs), 9 CD36-sensory neuron membrane proteins (CD36/SNMPs), 4 chemosensory proteins (CSPs), 4 myeloid lipid-recognition (ML) and Niemann-Pick C2 disease proteins (ML/NPC2), 2 pickpocket receptors (PPKs) and 3 transient receptor potential channels (TRPs). Differential expression analyses identified sex-biased sensory receptors.ConclusionsWe identified sensory receptors that were differentially expressed between the antennae of both sexes and hence may be associated with host detection by female flies. The most promising for future investigations were as follows: an odorant receptor (LcupOR46) which is female-biased in L. cuprina and Cochliomyia hominivorax Coquerel, 1858; an ABC transporter (ABC G23.1) that was the sole sensory receptor upregulated in the antennae of females attracted to rotting beef compared to non-attracted females; a female-biased ammonia transporter (AMT_Rh50), which was previously associated with ammonium detection in Drosophila melanogaster Meigen, 1830. This is the first report suggesting a possible role for ABC transporters in L. cuprina olfaction and potentially in other insects.Graphical

A Female-Biased Chemosensory Protein PxutCSP19 in the Antennae of Papilio xuthus Tuned 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.

Transcriptome and differential expression analysis of chemosensory genes in the antennae and heads of Apis cerana cerana drones

The olfactory system plays a critical role in honey bee adaptation to complex and constantly changing environments, as well as in the maintenance of social cohesion. Honey bees have evolved a sophisticated chemical communication system to regulate their intricate social interactions. Despite there is a preliminary understanding of the components, cells, and physiological pathways involved in pheromone response, the molecular mechanisms underlying honey bee drones’ mating behavior and pheromone communication remain largely unknown. This study presented the first comprehensive transcriptome analysis of the antennae and heads of Apis cerana cerana drones in response to 9-ODA. Based on gene ontology and KEGG pathway, we found the majority of differentially expressed genes associated with molecular functions and signal transduction. Additionally, we identified 153 genes closely related to olfaction in the transcriptome datasets, including 111 odorant receptors (ORs), 16 odorant-binding proteins (OBPs), nine ionotropic receptors (IRs), 10 gustatory receptors (GRs), five chemosensory proteins (CSPs), and two sensory neuron membrane proteins (SNMPs), and most of them showed relatively high expression levels in the drone antennae. The reliability of these differential genes was validated using RT-qPCR. Our results will contribute to detect the molecular mechanisms of odorant and investigate olfactory functions in the Asian honey bees.

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 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.

Identification of chemosensory genes and antennal sensilla in Nassophasis sp. (Coleoptera: Rhynchophorinae)

Phytophagous insects rely on plant volatiles to select and locate hosts for feeding or reproduction and their olfactory system is essential for detecting plant volatiles. The stem-boring pest, Nassophasis sp. damages Dendrobium and causes economic losses. Currently, there are no effective methods for its control. However, understanding the morphological and molecular basis of its olfactory system may identify new pathways for their management and control. In this study, we observed the stemborer's antennal sensilla using scanning electron microscopy, and transcriptome sequencing was undertaken to annotate and analyze its chemosensory genes. Results showed that the antennal morphology is similar between males and females, with five types of antennal sensilla observed: sensilla chaetica (SC), sensilla trichodea (ST), sensilla brush (SB), sensilla basiconica (SBA) and sensilla gemmiformium (SG). Sexual dimorphism was not observed in sensilla type, but in the length of SBA and SG. A total of 70 olfactory-related genes were annotated, including 16 odorant binding proteins (OBP), 5 chemosensory proteins (CSPs), 26 olfactory receptors (ORs), 9 gustatory receptors (GRs), 10 ionotropic receptors (IRs), and 4 sensory neuron membrane proteins (SNMPs). Most genes were highly expressed and 14 of these genes were only expressed in the head, and 7 genes in the abdomen. This study provides a theoretical basis for the olfactory perception of Nassophasis sp. and a scientific basis for developing new pest control strategies.

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.

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.