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Abstract Background Endoclita signifer causes severe damage to eucalyptus plantations, and the larvae transfer to and damage eucalyptus accurately in mixed forests, suggesting that the larval olfactory system contributes to host selection. The olfactory proteins in the head and tegument of E. signifer larvae were previously identified. To identify the relationship between olfactory protein expression in the larval head the larvae head and the developmental expression dynamics, and its functions in further recognition of plant volatiles, the head transcriptomes of two instar larvae and the expression profiles of olfactory proteins in the instars after exposure to volatiles were studied. Results Eight odorant-binding proteins, six chemosensory proteins, three odorant receptors, three gustatory receptors, and 18 ionotropic receptors were identified. Half of the olfactory proteins were the most highly expressed in the young (5th) larval head, and EsigGOBP2, EsigGOBP4, EsigGOBP5, EsigCSP1, EsigCSP3, EsigGR1 and EsigGR3 were highly expressed and showed a specific expression pattern. In addition, after exposure to o-cymene, α-phellandrene, n-butyl ether, and 4-ethylacetophenone, EsigGR3 was downregulated significantly, and exposure to n-butyl ether caused EsigGR1 to be downregulated significantly. Conclusions Seven specific olfactory proteins may be important genes in larval olfactory recognition. Furthermore, based on the receptors that were downregulated after exposure to volatiles and the previous electrophysiological activity in the third larvae, we speculated that the ligand of EsigGR1 was n-butyl ether, and the ligands of the newly identified EsigGR3 are all electrophysiologically active compounds, which demonstrated host recognition in the third larvae of E. signifer. These results provide a way to find key plant volatiles recognized by the key olfactory proteins as new targets for pest control. Graphical Abstract

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 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.

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.

BACKGROUND: Endoclita signifer is a polyphagous lepidopteran species of eucalyptus that selects its hosts in the third-instar larval period. To understand how it adapts to its host during development, we studied the olfactory responses of late-stage (fifth)-instar larvae to the dynamic chemical environment they encounter. RESULTS: Thirty-two volatiles from eucalyptus trunk and soil were identified, among which 14 showed electroantennal activity and five were identified as new. Further behavioral bioassay showed that both β-pinene and the imitation ratio of six key volatile in eucalyptus trunk were attractive to the fifth-instar larvae, but both eliminated and increased β-pinene in the mixture decreased the choice ratio and showed no attraction. Although E. signifer larvae shifted their attraction from o-cymene at the third-instar stage to β-pinene at the fifth-instar stage in a single volatile, the appropriate ratio of the main compounds in eucalyptus trunk volatile is the key to the behavior choice of fifth-instar larvae. CONCLUSION: The switch in olfactory attraction to different compounds between fifth- and third-instar larvae indicated an olfactory plasticity between third- and fifth-instar larvae. And the particular blend mediated the highly specialized communication interactions specificity between fifth-instar larvae and eucalyptus trunk volatile indicated the specialized host adaptation to fifth-instar larvae. This enhances understanding of how the primitive lepidopteran E. signifier, as a native pest, adapts to introduced eucalyptus. Moreover, this study provides knowledge for the screening and development of target volatiles for trapping and managing E. signifer larvae. [ABSTRACT FROM AUTHOR]