Eogystia hippophaecolus (Hua et al.) (Lepidoptera: Cossidae) is the major threat to seabuckthorn plantations in China. Specific and highly efficient artificial sex pheromone traps was developed and used to control it. However, the molecular basis for the pheromone recognition is not known. So we established the antennal transcriptome of E. hippophaecolus and characterized the expression profiles of odorant binding proteins. These results establish and improve the basis knowledge of the olfactory receptive system, furthermore provide a theoretical basis for the development of new pest control method. We identified 29 transcripts encoding putative odorant-binding proteins (OBPs), 18 putative chemosensory proteins (CSPs), 63 odorant receptors (ORs), 13 gustatory receptors (GRs), 12 ionotropic receptors (IRs), and two sensory neuron membrane proteins (SNMPs). Based on phylogenetic analysis, we found one Orco and three pheromone receptors of E. hippophaecolus and found that EhipGR13 detects sugar, EhipGR11 and EhipGR3 detect bitter. Nine OBPs expression profile indicated that most were the highest expression in antennae, consistent with functions of OBPs in binding and transporting odors during the antennal recognition process. OBP6 was external expressed in male genital-biased in, and this locus may be responsible for pheromone binding and recognition as well as mating. OBP1 was the highest and biased expressed in the foot and may function as identification of host plant volatiles. One hundred thirty-seven chemosensory proteins were identified and the accurate functions and groups of part proteins were obtained by phylogenetic analysis. The most OBPs were antenna-biased expressed, which are involved in antennal recognition. However, few OBP was detected biased expression in the foot and external genitalia, and these loci may function in pheromone recognition, mating, and the recognition of plant volatiles.

Leptocybe invasa (Hymenoptera: Eulophidae) is a globally intrusive pest. Despite extensive research into the physiological responses of this pest, our understanding of the molecular mechanisms still needs to be improved. We want to accurately investigate the expression of L. invasa’s target genes, so it is imperative to select fitting reference genes. In this study, eight housekeeping genes’ stability (RPS30, ACTR, 18S rRNA, ACT, RPL18, GAPDH, 28S rRNA, and TUB) was tested under five different experimental conditions, including male or female adults, somites (head, thorax, and abdomen), temperatures (0 °C, 25 °C, and 40 °C), diets (starvation, clear water, 10% honey water, Eucalyptus sap), and pesticides (acetone was used as a control, imidacloprid, monosultap). Gene stability was calculated using RefFinder, which integrates four algorithms (the ∆Ct method, geNorm, NormFinder, and BestKeeper). The findings implied that ACT and ACTR were the most accurate when comparing sexes. For analyzing different somites, 28S rRNA and RPL18 were ideal; the 28S rRNA and RRS30 were perfect for analyzing at different temperatures. The combination of ACT and GAPDH helped to analyze gene expression in different diets, and GAPDH and 28S rRNA were suitable for various pesticide conditions. Overall, this research offers a complete list of reference genes from L. invasa for precise analysis of target gene expression, which can improve the trustworthiness of RT-qPCR and lay the foundation for further investigations into the gene function of this pest.

Endoclita signifier Walker (Lepidoptera: Hepialidae), a polyphagous insect, has become a new wood-boring pest in Eucalyptus plantations in southern China since 2007, which represents a typical example of native insect adaptation to an exotic host. After the third instar, larvae move from soil to standing trees and damage the plants with a wormhole. Although females disperse to lay eggs, larvae can accurately find eucalyptus in a mingled forest of eight species, which leads us to hypothesize that the larval olfactory system contributes to its host selection. Herein, we investigated the transcriptomes of the head and tegument of E. signifer larvae and explored the expression profiles of olfactory proteins. We identified 15 odorant-binding proteins (OBPs), including seven general OBPs (GOPBs), six chemosensory proteins (CSPs), two odorant receptors (ORs), one gustatory receptor (GR), 14 ionotropic receptors (IRs), and one sensory neuron membrane protein (SNMP). Expression profiles indicated that all olfactory proteins, except for EsigCSP1, were expressed in the head, and most were also detected in non-olfactory tissues, especially thorax tegument. Furthermore, EsigOBP2, EsigOBP8, EsigGOBP1, EsigGOBP2, EsigGOBP5, EsigCSP3, EsigCSP5, and EsigOR1 were expressed most strongly in the head; moreover, EsigCSP3 expressed abundantly in the head. EsigGR1 exhibited the highest expression among all tissues. Besides phylogenetic analysis shows that EsigGOBP7 probably is the pheromone-binding protein (PBP) of E. signifier. This study provides the molecular basis for future study of chemosensation in E. signifier larvae. EsigCSP3 and EsigGR1, which have unique expression patterns, might be factors that govern the host choice of larvae and worth further exploration.

Endoclita signifer is a major wood-boring pest of eucalyptus trees in China, and its third instar larvae can accurately find and infest eucalyptus trees in mixed-species forests, although it can also feed on another 51 tree species in the same planted area. A total of 34 volatile compounds were identified from eucalyptus trunks, including non-infested and infested trunks with single or multiple (more than three) larval entrance cover packets, and forest floor humus. Of these, o-cymene showed a strong gas chromatography-electroantennographic detection (GC-EAD) activity and significant attraction of the third instar E. signifer larvae. Higher abundance of alpha-phellandrene, o-cymene, and the unique 2-phenyl-2-propanol in the volatile profile from infested eucalyptus trunks indicated that they were herbivore-induced plant volatiles (HIPVs). The larvae showed significantly higher attraction to volatile blends, especially those from infested eucalyptus trunks, than to single volatiles. A blend of the three HIPVs, α-pinene, D-limonene, and n-butyl ether may serve as an attractant for the control of E. signifer larvae in eucalyptus plantations. Further, exposure of third instar E. signifer larvae to some of these volatiles which also elicited electroantennogram and behavioral responses, influenced expressions of some olfactory proteins. Our results show that third instar E. signifer larvae can recognize o-cymene from host eucalyptus trunks and are attracted to the trunk by the three HIPVs when they shift their habitats from the forest floor humus to the tree trunks.

Pheromone-binding proteins (PBPs) are specific odorant-binding proteins that can specifically recognize insect pheromones. Through transcriptional analysis of the antennae of adult Endoclita signifer, EsigPBP3 was discovered and identified, and EsigPBP3 was found to be highly expressed in the antennae of male moths. Based on the binding characteristics and ability of EsigPBP3, we can find the key ligands and binding site to consider as a target to control the key wood bore E. signifier. In this study, the fluorescence competitive binding assays (FCBA) showed that EsigPBP3 had a high binding affinity for seven key eucalyptus volatiles. Molecular docking analysis revealed that EsigPBP3 had the strongest binding affinity for the sexual pheromone component, (3E,7E)-4,7,11-trimethyl-1,3,7,10-dodecatetraene. Furthermore, same as the result of FCBA, the EsigPBP3 exhibited high binding affinities to key eucalyptus volatiles, eucalyptol, α-terpinene, (E)-beta-ocimene, (−)-β-pinene, and (−)-α-pinene, and PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 are key sites. In summary, EsigPBP3 exhibits high binding affinity to male pheromones and key volatile compounds and the crucial binding sites PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 can act as targets in the recognition of E. signifier pheromones.