Expression Pattern and Ligand Binding Characteristics Analysis of Chemosensory Protein SnitCSP2 from Sirex nitobei.

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.

Investigating SnocCSP4 expression and key compound interactions with SnocOBP4 in Sirex noctilio Fabricius (Hymenoptera: Siricidae).

Sirex noctilio, a significant pest impacting Pinus sylvestris var. mongolica, presents control difficulties due to its wood-boring behavior, paucity of natural antagonists, and wide-ranging habitats. Our research aims to elucidate the functionality and operational mechanisms of chemosensory proteins 4 (SnocCSP4), providing strategic insights for pest management and fostering further exploration in CSPs. Techniques such as qPCR, fluorescence binding affinity assays, molecular docking, and dynamic simulations were utilized to investigate the tissue-specific distribution, ligand binding capacities, and mechanistic underpinnings of SnocCSP4. The findings revealed a high abundance of SnocCSP4 in male genitalia, significant sexual dimorphism in its expression, and high binding affinities to (-)-Globulol and 10-Oxodecanoic acid. Subsequent analysis identified hydrophobic cavities formed by non-polar amino acids (VAL, LEU, ILE, LYS) and the critical role of polar amino acids (ALA 46, GLU 45, THR 75) in maintaining system stability. These insights suggest the primary role of SnocCSP4 in binding or transporting these volatiles and indicate that modifying key amino acids could inform the design of more effective pest control measures.

Morphology and location of sensilla in the antennae and ovipositor of Sirex noctilio (Hymenoptera: Siricidae).

The woodwasp Sirex noctilio Fabricius is a major quarantine pest that was reported in China in 2013 and mostly damages Pinus sylvestris var. mongolica. Reverse chemical ecology, which uses chemical lures to catch or block insects from mating is the classic way to control forestry pests. This indicates that insect sensilla play a crucial role in detecting external chemical and physical stimuli. Nonetheless, the categorization and distribution of sensilla on the antennae and ovipositor of S. noctilio are insufficiently specific. In this paper, scanning electron microscopy (SEM) was used to observe the ultrastructure of the sensilla of S. noctilio on the antenna and ovipositor. It was found that the types and distribution of sensilla on the antennae of S. noctilio male and female are consistent, and six types of sensilla are found: sensilla trichodea (ST), sensilla chaetica (SC), Böhm bristles (BB), sensilla basiconica (SB), sensilla ampullacea (SA), and contact chemoreceptors (CC). Besides, there are five types of sensilla on the female ovipositor. In addition to ST, SC and BB, two more types of sensilla are also found: sensilla cavity (SCa) and sensilla coeloconica (SCo). Through identification of the morphology and distribution of the sensilla, the functions of different sensilla in the mating and host selection mechanisms of S. noctilio are proposed, thereby establishing a foundation for S. noctilio chemical communication research.

Mating Behavior and Identification of Male-Produced Pheromone Components in Two Woodwasps, Sirex noctilio and Sirex nitobei, in China.

To protect vulnerable trees from native and invasive wood wasps, the mating behavior of these two woodwasp species (S. noctilio and S. nitobei, respectively) and factors influencing this behavior were investigated in cages outdoors. Male-produced pheromones were identified in both woodwasp species. Compared with the native species S. nitobei, the invasive species S. noctilio showed stronger mating ability, including mating frequency, time, and duration. The mating behavior of both species mainly occurred from 9:00 to 17:00 each day, peaking at 11:00 and 12:00. The daily mating behavior of both species was most directly related to light intensity. Both female and male S. noctilio and S. nitobei were capable of mating upon emergence, and most individuals mated at 2 days of age. For both species, a female-to-male ratio of 5:15 was most conducive to mating, and individuals with a larger body size were preferred as mates by males and females. (Z)-3-decenol was present in solid-phase microextraction extracts of both species. Two reported minor reference components, (Z)-4-decen-1-ol and (E, E)-2,4-decadienal, were not identified in either woodwasp species. The peak of male pheromone release occurred from 11:00-12:00 for 2-day-old individuals.

Key Residues Affecting Binding Affinity of Sirex noctilio Fabricius Odorant-Binding Protein (SnocOBP9) to Aggregation Pheromone.

Sirex noctilio Fabricius (Hymenoptera Siricidae) is a major quarantine pest responsible for substantial economic losses in the pine industry. To achieve better pest control, (Z)-3-decen-ol was identified as the male pheromone and used as a field chemical trapping agent. However, the interactions between odorant-binding proteins (OBPs) and pheromones are poorly described. In this study, SnocOBP9 had a higher binding affinity with Z3D (Ki = 1.53 ± 0.09 µM) than other chemical ligands. Molecular dynamics simulation and binding mode analysis revealed that several nonpolar residues were the main drivers for hydrophobic interactions between SnocOBP9 and Z3D. Additionally, computational alanine scanning results indicated that five amino acids (MET54, PHE57, PHE71, PHE74, LEU116) in SnocOBP9 could potentially alter the binding affinity to Z3D. Finally, we used single-site-directed mutagenesis to substitute these five residues with alanine. These results imply that the five residues play crucial roles in the SnocOBP9-Z3D complex. Our research confirmed the function of SnocOBP9, uncovered the key residues involved in SnocOBP9-Z3D interactions, and provides an inspiration to improve the effects of pheromone agent traps.

EsigGOBP1: The Key Protein Binding Alpha-Phellandrene in Endoclita signifer Larvae.

Endoclita signifer larvae show olfactory recognition towards volatiles of eucalyptus trunks and humus soils. Further, EsigGOBP1 was identified through larval head transcriptome and speculated as the main odorant-binding proteins in E. signifer larvae. In this study, the highest expression of EsigGOBP1 was only expressed in the heads of 3rd instar larvae of E. signifer, compared with the thorax and abdomen; this was consistent with the phenomenon of habitat transfer of 3rd instar larvae, indicating that EsigGOBP1 was a key OBP gene in E. signifer larvae. Results of fluorescence competition binding assays (FCBA) showed that EsigGOBP1 had high binding affinities to eight GC-EAD active ligands. Furthermore, screening of key active odorants for EsigGOBP1 and molecular docking analysis, indicated that EsigGOBP1 showed high binding activity to alpha-phellandrene in 3rd instar larvae of E. signifer. Conformational analysis of the EsigGOBP1-alpha-phellandrene complex, showed that MET49 and GLU38 were the key sites involved in binding. These results demonstrated that EsigGOBP1 is a key odorant-binding protein in E. signifer larvae, which recognizes and transports eight key volatiles from eucalyptus trunk, especially the main eucalyptus trunks volatile, alpha-phellandrene. Taken together, our results showed that EsigGOBP1 is involved in host selection of E. signifer larvae, which would aid in developing EsigGOBP1 as molecular targets for controlling pests at the larval stage.

Antennal transcriptome analysis of olfactory genes and tissue expression profiling of odorant binding proteins in Semanotus bifasciatus (cerambycidae: coleoptera).

BACKGROUND: Insect olfactory proteins can transmit chemical signals in the environment that serve as the basis for foraging, mate searching, predator avoidance and oviposition selection. Semanotus bifasciatus is an important destructive borer pest, but its olfactory mechanism is not clear. We identified the chemosensory genes of S. bifasciatus in China, then we conducted a phylogenetic analysis of the olfactory genes of S. bifasciatus and other species. And the expression profiles of odorant binding proteins (OBPs) genes in different tissues and different genders of S. bifasciatus were determined by quantitative real-time PCR for the first time. RESULTS: A total of 32 OBPs, 8 chemosensory proteins (CSPs), 71 odorant receptors (ORs), 34 gustatory receptors (GRs), 18 ionotropic receptors (IRs), and 3 sensory neuron membrane proteins (SNMPs) were identified. In the tissue expression analysis of OBP genes, 7 OBPs were higher expressed in antennae, among them, SbifOBP2, SbifOBP3, SbifOBP6, SbifOBP7 and SbifOBP20 were female-biased expression, while SbifOBP1 was male-biased expression and SbifOBP22 was no-biased expression in antennae. In addition, the expressed levels of SbifOBP4, SbifOBP12, SbifOBP15, SbifOBP27 and SbifOBP29 were very poor in the antennae, and SbifOBP4 and SbifOBP29 was abundant in the head or legs, and both of them were male-biased expression. While SbifOBP15 was highly expressed only at the end of the abdomen with its expression level in females three times than males. Other OBPs were expressed not only in antennae but also in various tissues. CONCLUSION: We identified 166 olfactory genes from S. bifasciatus, and classified these genes into groups and predicted their functions by phylogenetic analysis. The majority of OBPs were antenna-biased expressed, which are involved in odor recognition, sex pheromone detection, and/or host plant volatile detection. However, also some OBPs were detected biased expression in the head, legs or end of the abdomen, indicating that they may function in the different physiological processes in S. bifasciatus.

Antennal transcriptome analysis of olfactory genes and characterizations of odorant binding proteins in two woodwasps, Sirex noctilio and Sirex nitobei (Hymenoptera: Siricidae).

BACKGROUND: The woodwasp Sirex noctilio Fabricius is a major quarantine pest worldwide that was first discovered in China in 2013 and mainly harms Pinus sylvestris var. mongolica Litv.. S. nitobei Matsumura is a native species in China and is closely related to S. noctilio. Recently, the two woodwasps species were found attacking the P. sylvestris var. mongolica Litv in succession. The olfactory system is the foundation of insect behavior. Olfactory genes were identified through antennal transcriptome analysis. The expression profiles odorant binding proteins (OBPs) were analyzed with RT-qPCR. RESULTS: From our transcriptome analysis, 16 OBPs, 7 chemosensory proteins (CSPs), 41 odorant receptors (ORs), 8 gustatory receptors (GRs), 13 ionotropic receptors (IRs), and one sensory neuron membrane protein (SNMP) were identified in S. noctilio, while 15 OBPs, 6 CSPs, 43 ORs, 10 GRs, 16 IRs, and 1 SNMP were identified in S. nitobei. Most of the olfactory genes identified in two species were homologous. However, some species-specific olfactory genes were identified from the antennal transcriptomes, including SnocOBP13, SnocCSP6, SnocOR26, SnocGR2, SnocIR7 in S. noctilio and SnitGR9, SnitGR11, SnitIR17 in S. nitobei. In total, 14 OBPs were expressed primarily in the antennae. SnocOBP9 and SnitOBP9, identified as PBP homologues, were sex-biased expression in two siricid, but with different pattern. SnocOBP11 and SnitOBP11 were highly expressed in antennae and clearly expressed in external genitalia. SnocOBP7 and SnitOBP7 were highly expressed in male genitalia. SnocOBP3 and SnocOBP10 were highly expressed in female genitalia and male heads, while SnitOBP3 and SnitOBP10 did not show obvious tissue bias. CONCLUSION: We analyzed 86 and 91 olfactory genes from S. noctilio and S. nitobei, respectively. Most of the olfactory genes identified were homologous, but also some species-specific olfactory genes were identified, which indicated the similarities and differences of the molecular mechanisms between the two closely-related species. Different expression in the antennae, external genitals or heads, exhibiting an obvious sex bias, suggested their different role in recognizing sex pheromones or plant volatiles. Species-specific expression for several OBPs genes may suggest that they strengthened or lost their original function during species differentiation, resulting in olfactory differences between the two species.